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1.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献
2.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
3.
Zhemin Zhou Yoshiteru Hashimoto Michihiko Kobayashi 《The Journal of biological chemistry》2009,284(22):14930-14938
4.
5.
Leonard Kaysser Liane Lutsch Stefanie Siebenberg Emmanuel Wemakor Bernd Kammerer Bertolt Gust 《The Journal of biological chemistry》2009,284(22):14987-14996
Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics
isolated from Streptomyces sp. MK730-62F2 and belong to the
translocase I inhibitor family. Their complex structure is derived from
5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique
N-methyldiazepanone ring. The biosynthetic gene cluster has been
identified, cloned, and sequenced, representing the first gene cluster of a
translocase I inhibitor. Sequence analysis revealed the presence of 23 open
reading frames putatively involved in export, resistance, regulation, and
biosynthesis of the caprazamycins. Heterologous expression of the gene cluster
in Streptomyces coelicolor M512 led to the production of
non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions
validated the boundaries of the cluster and inactivation of cpz21
resulted in the accumulation of novel simplified liponucleoside antibiotics
that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as
an acyltransferase in caprazamycin biosynthesis. In vivo and in
silico analysis of the caprazamycin biosynthetic gene cluster allows a
first proposal of the biosynthetic pathway and provides insights into the
biosynthesis of related uridyl-antibiotics.Caprazamycins
(CPZs)2
(Fig. 1, 1) are
liponucleoside antibiotics isolated from a fermentation broth of
Streptomyces sp. MK730-62F2
(1,
2). They show excellent
activity in vitro against Gram-positive bacteria, in particular
against the genus Mycobacterium including Mycobacterium
intracellulare, Mycobacterium avium, and Mycobacterium
tuberculosis (3). In a
pulmonary mouse model with M. tuberculosis H37Rv, administration of
caprazamycin B exhibited a therapeutic effect but no significant toxicity
(4). Structural elucidation
(2) revealed a complex and
unique composition of elements the CPZs share only with the closely related
liposidomycins (LPMs, 2)
(5). The core skeleton is the
(+)-caprazol (5)
composed of an N-alkylated
5′-(β-O-aminoribosyl)-glycyluridine, also known from
FR-900493 (6)
(6) and the muraymycins
(7)
(7), which is cyclized to form
a rare diazepanone ring. Attached to the 3′″-OH are β-hydroxy
fatty acids of different chain length resulting in CPZs A–G
(1). They differ from
the LPMs in the absence of a sulfate group at the 2″-position of the
aminoribose and the presence of a permethylated l-rhamnose
β-glycosidically linked to the 3-methylglutaryl (3-MG) moiety.Open in a separate windowFIGURE 1.Nucleoside antibiotics of the translocase I inhibitor family.The LPMs have been shown to inhibit biosynthesis of the bacterial cell wall
by targeting the formation of lipid I
(8). The CPZs are expected to
act in the same way and are assigned to the growing number of translocase I
inhibitors that include other nucleoside antibiotics, like the tunicamycins
and mureidomycins (9). During
peptidoglycan formation, translocase I catalyzes the transfer of
UDP-MurNAc-pentapeptide to the undecaprenyl phosphate carrier to
generate lipid I (10). This
reaction is considered an unexploited and promising target for new
anti-infective drugs (11).Recent investigations indicate that the 3″-OH group
(12), the amino group of the
aminoribosyl-glycyluridine, and an intact uracil moiety
(13) are essential for the
inhibition of the Escherichia coli translocase I MraY. The chemical
synthesis of the (+)-caprazol
(5) was recently
accomplished (14), however,
this compound only shows weak antibacterial activity. In contrast, the
acylated compounds 3 and 4 exhibit strong growth inhibition of
mycobacteria, suggesting a potential role of the fatty acid side chain in
penetration of the bacterial cell
(15,
16). Apparently, the
acyl-caprazols (4)
represent the most simplified antibiotically active liponucleosides and a good
starting point for further optimization of this class of potential
therapeutics.Although chemical synthesis and biological activity of CPZs and LPMs has
been studied in some detail, their biosynthesis remains speculative and only
few data exists about the formation of other translocase I inhibitors
(17,
18). Nevertheless, we assume
that the CPZ biosynthetic pathway is partially similar to that of LPMs,
FR-90043 (6), and
muraymycins (7) and
presents a model for the comprehension and manipulation of liponucleoside
formation. Considering the unique structural features of the CPZs we also
expect some unusual biotransformations to be involved in the formation of,
e.g. the (+)-caprazol.Here we report the identification and analysis of the CPZ gene cluster, the
first cluster of a translocase I inhibitor. A set of gene disruption
experiments provide insights into the biosynthetic origin of the CPZs and
moreover, heterologous expression of the gene cluster allows the generation of
novel bioactive derivatives by pathway engineering. 相似文献
6.
Joseph T. Roland Lynne A. Lapierre James R. Goldenring 《The Journal of biological chemistry》2009,284(2):1213-1223
Rab proteins influence vesicle trafficking pathways through the assembly of
regulatory protein complexes. Previous investigations have documented that
Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate
distinct trafficking pathways. We have now determined that a related Rab
protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10
localized to a system of tubules and vesicles that have partially overlapping
localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the
expression of dominant-negative myosin V tails. Interaction with Rab10 was
dependent on the presence of the alternatively spliced exon D in myosin Va and
myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and
fluorescence resonance energy transfer studies confirmed that Rab10 binding to
myosin V tails in vivo required the alternatively spliced exon D. In
contrast to our previous work, we found that Rab11a can interact with both
myosin Va and myosin Vb tails independent of their splice isoform. These
results indicate that Rab GTPases regulate diverse endocytic trafficking
pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous
structures that require the efficient and timely movement of diverse
intracellular proteins for proper function. Molecular motors provide the
physical force needed to move these materials along microtubules and actin
microfilaments. Unconventional myosin motors, such as those belonging to
classes V, VI, and VII, have roles in the trafficking and recycling of
membrane-bound structures in eukaryotic cells
(1) and are recruited to
discrete vesicle populations. Myosin VI is involved in clathrin-mediated
endocytosis (2), whereas myosin
VIIa participates in the proper development of stereocilia of inner ear hair
cells and the transport of pigment granules in retinal pigmented epithelial
cells (3,
4). Similarly, the three
members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc,
are required for the proper transport of a wide array of membrane cargoes,
such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical
recycling endosomes in polarized epithelial cells, and bulk recycling vesicles
in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems,
including membrane trafficking
(6,
7). Certain Rab proteins
associate with and regulate the function of class V myosins. Rab27a, in a
complex with the adaptor protein melanophilin/Slac2-a, is required to localize
myosin Va to the surface of melanin-filled pigment granules in vertebrates
(8-10),
whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa
(3,
11). Rab11a, in a complex with
its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling
endosomes
(12-14)
where the tripartite complex regulates the recycling of a variety of cargoes
(15-19).
In addition, Rab8a associates with both myosin Vb
(20) and myosin Vc
(21) as part of the
non-clathrin-mediated tubular recycling system
(20). Recently, Rab11a has
also been shown to associate with myosin Va in the transport of AMPA receptors
in dendritic spines (22),
contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va
in a tissue-specific manner
(23-28).
Alternate splicing occurs in a region lying between the coiled-coil region of
the neck of the motor and the globular tail region. Three exons in particular
are subject to alternative splicing: exons B, D, and F
(23-25).
Exon F is critical for association with melanophilin/Slac2 and Rab27a
(8,
9,
29,
30). Additionally, exon B is
required for the interaction of myosin Va with dynein light chain 2 (DLC2)
(27,
28). Currently no function for
the alternatively spliced exon D has been reported. Similar to myosin Va,
myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been
identified in myosin Vb (Fig.
1A). In addition, exon B in myosin Vb does not resemble
the dynein light chain 2 (DLC2) binding region in myosin Va
(27,
28), and therefore, it likely
does not interact with DLC2. On the other hand, exon D is highly conserved
among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in
these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice
isoforms. A, schematic of the alternative exon organization in
the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are
subject to alternative splicing in myosin Va, whereas there is only evidence
that exon D is alternatively spliced in myosin Vb, which does not contain exon
F. B, alignment of exon D sequences from mouse and human myosin V''s.
myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin
Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like
region (amino acids 1124-1147 of human myosin Vc) that is not known to be
alternatively spliced. Alignment of the exon D regions from all three motors
reveals a high degree of homology, especially in the center of the exon.
Asterisks indicate amino acid identities. C, PCR-based
analysis of human tissue panels reveals the alternative splicing pattern of
exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D
for both motors were used to amplify cDNA from human MTC™ panels
(Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin
Vb tail constructs were used as positive controls. Variants expressing exon D
(upper bands) and lacking exon D (lower bands) were visible.
Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have
similar function
(31-35),
localizes to a system of tubules and vesicles overlapping in distribution with
Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we
show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin
Vc in vivo. In addition, we have determined that the alternatively
spliced exon D in both myosin Va and myosin Vb is required for interaction
with Rab10. In contrast to our previous findings, we demonstrate that Rab11a
is able to interact with both myosin Va and myosin Vb tails in an exon
independent-manner. These results reveal that multiple Rab proteins
potentially regulate all three class V myosin motors. 相似文献
7.
Jeffrey M. Boyd Jamie L. Sondelski Diana M. Downs 《The Journal of biological chemistry》2009,284(1):110-118
The ApbC protein has been shown previously to bind and rapidly transfer
iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J.,
Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47,
8195–8202. This study utilized both in vivo and in
vitro assays to examine the function of variant ApbC proteins. The in
vivo assays assessed the ability of ApbC proteins to function in pathways
with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins
were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S]
cluster, and transfer [Fe-S] cluster. This study details the first kinetic
analysis of ATP hydrolysis for a member of the ParA subfamily of
“deviant” Walker A proteins. Moreover, this study details the
first functional analysis of mutant variants of the ever expanding family of
ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that
ApbC protein needs ATPase activity and the ability to bind and rapidly
transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide
array of metabolic functions (reviewed in Ref.
1). Research addressing the
biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter
vinelandii led to the discovery of an operon
(iscAnifnifUSVcysE1) involved in the
biosynthesis of [Fe-S] clusters (reviewed in Ref.
2). Subsequent experiments led
to the finding of two more systems involved in the de novo
biosynthesis of [Fe-S] clusters, the isc and the suf systems
(3,
4). Like Escherichia
coli, the genome of Salmonella enterica serovar Typhimurium
encodes for the isc and suf [Fe-S] cluster biosynthesis
machinery.Recent studies have identified a number of additional or
non-isc/-suf-encoded proteins that are involved in bacterial
[Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY,
an iron-binding protein believed to be involved in iron trafficking and iron
delivery
(5–7);
YggX, an Fe2+-binding protein that protects the cell from oxidative
stress (8,
9); ErpA, an alternate A-type
[Fe-S] cluster scaffolding protein
(10); NfuA, a proposed
intermediate [Fe-S] delivery protein
(11–13);
YtfE, a protein proposed to be involved in [Fe-S] cluster repair
(14,
15); and CsdA-CsdE, an
alternative cysteine desulferase
(16).Analysis of the metabolic network anchored to thiamine biosynthesis in
S. enterica identified lesions in three non-isc or
-suf loci that compromise Fe-S metabolism as follows: apbC,
apbE, and rseC
(17–21).
This metabolic system was subsequently used to dissect a role for
cyaY and gshA in [Fe-S] cluster metabolism
(6,
22,
23). Of these, the
apbC (mrp in E. coli) locus was identified as the
predominant site of lesions that altered thiamine synthesis by disrupting
[Fe-S] cluster metabolism (17,
18).ApbC is a member of the ParA subfamily of proteins that have a wide array
of functions, including electron transfer
(24), initiation of cell
division (25), and DNA
segregation (26,
27). Importantly, ATP
hydrolysis is required for function of all well characterized members of this
subfamily, and all members contain a “deviant” Walker A motif,
which contains two lysine residues instead of one (GKXXXGK(S/T))
(28). ApbC has been shown to
hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been
shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence
alignments of the central portion of these proteins and bacterial ApbC are
shown in Fig. 1. HCF101 was
demonstrated to be involved in chloroplast [Fe-S] cluster metabolism
(29,
30). The CFD1, Npb35, and
huNbp35 (formally Nubp1) proteins were demonstrated to be involved in
cytoplasmic [Fe-S] cluster metabolism
(31,
32). Ind1 was demonstrated to
be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme
NADH:ubiquinone oxidoreductase
(33). There is currently no
report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of
ParA family of proteins. Protein alignments were assembled using the
Clustal_W method in the Lasergene® software and show only the central
portion of the proteins, which have the highest sequence conservation. The
three boxed areas highlight the Walker A box, conserved Ser residue,
and CXXC motif. Proteins listed are as follows: ApbC (S.
enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35
(S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35
(formally Nubp1) (Homo sapiens), and Ind1 (Candida
albicans).Biochemical analysis of ApbC indicated that it could bind and transfer
[Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate
isomerase (34). Additional
genetic studies indicated that ApbC has a degree of functional redundancy with
IscU, a known [Fe-S] cluster scaffolding protein
(35,
36).In this study we investigate the correlation between the biochemical
properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and
[Fe-S] cluster transfer rates) and the in vivo function of this
protein. This is the first detailed kinetic analysis of ATP hydrolysis for a
member of the ParA subfamily of deviant Walker A proteins and the first
functional analysis of a member of the ever expanding family of ApbC/Nbp35
proteins. Data presented indicate that noncomplementing variants have distinct
biochemical properties that place them in three distinct classes. 相似文献
8.
Bryson W. Katona Shrikant Anant Douglas F. Covey William F. Stenson 《The Journal of biological chemistry》2009,284(5):3354-3364
Bile acids are steroid detergents that are toxic to mammalian cells at high
concentrations; increased exposure to these steroids is pertinent in the
pathogenesis of cholestatic disease and colon cancer. Understanding the
mechanisms of bile acid toxicity and apoptosis, which could include
nonspecific detergent effects and/or specific receptor activation, has
potential therapeutic significance. In this report we investigate the ability
of synthetic enantiomers of lithocholic acid (ent-LCA),
chenodeoxycholic acid (ent-CDCA), and deoxycholic acid
(ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116
cells. Natural bile acids were found to induce more apoptotic nuclear
morphology, cause increased cellular detachment, and lead to greater capase-3
and -9 cleavage compared with enantiomeric bile acids in both cell lines. In
contrast, natural and enantiomeric bile acids showed similar effects on
cellular proliferation. These data show that bile acid-induced apoptosis in
HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute
configuration of the bile steroid rather than its detergent properties. The
mechanism of LCA- and ent-LCA-induced apoptosis was also investigated
in HT-29 and HCT-116 cells. These bile acids differentially activate initiator
caspases-2 and -8 and induce cleavage of full-length Bid. LCA and
ent-LCA mediated apoptosis was inhibited by both pan-caspase and
selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor
provided no protection. LCA also induced increased CD95 localization to the
plasma membrane and generated increased reactive oxygen species compared with
ent-LCA. This suggests that LCA/ent-LCA induce apoptosis
enantioselectively through CD95 activation, likely because of increased
reactive oxygen species generation, with resulting procaspase-8 cleavage.Bile acids are physiologic steroids that are necessary for the proper
absorption of fats and fat-soluble vitamins. Their ability to aid in these
processes is largely due to their amphipathic nature and thus their ability to
act as detergents. Despite the beneficial effects, high concentrations of bile
acids are toxic to cells
(1-11).
High fat western diets induce extensive recirculation of the bile acid pool,
resulting in increased exposure of the colonic epithelial cells to these toxic
steroids (12,
13). A high fat diet is also a
risk factor for colon carcinogenesis; increased bile acid exposure is
responsible for some of this risk. Bile acids can contribute to both colon
cancer formation and progression, and their effects on colonic proliferation
and apoptosis aid this process by disrupting the balance between cell growth
and cell death, as well as helping to select for bile acid-resistant cells
(14,
15).In colonocyte-derived cell lines bile acid-induced apoptosis is thought to
proceed through mitochondrial destabilization with resulting mitochondrial
permeability transition formation and cytochrome c release as well as
generation of oxidative stress
(1,
9-11).
Bile acid-induced apoptosis has also been extensively explored in hepatocyte
derived cell lines with mechanisms including mitochondria dysfunction
(16-23),
endoplasmic reticulum stress
(24), ligand-independent
activation of death receptor pathways
(18,
25-28),
and modulation of cellular apoptotic and anti-apoptotic Bcl-2 family proteins
(29).Although ample evidence exists for multiple mechanisms of bile acid-induced
apoptosis, the precise interactions responsible for initiating these apoptotic
pathways are still unclear. Bile acids have been shown to interact directly
with specific receptors (30,
31). These steroids can also
initiate cellular signaling through nonspecific membrane perturbations
(32), and evidence exists
showing that other simple detergents (i.e. Triton X-100) are capable
of inducing caspase cleavage nonspecifically with resultant apoptosis
(33). Therefore, hydrophobic
bile acids may interact nonspecifically with cell membranes to alter their
physical properties, bind to receptors specific for these steroids, or utilize
a combination of both specific and nonspecific interactions to induce
apoptosis.Bile acid enantiomers could be useful tools for elucidating mechanisms of
bile acid toxicity and apoptosis. These enantiomers, known as
ent-bile acids, are synthetic nonsuperimposable mirror images of
natural bile acids with identical physical properties except for optical
rotation. Because bile acids are only made in one absolute configuration
naturally, ent-bile acids must be constructed using a total synthetic
approach. Recently we reported the first synthesis of three enantiomeric bile
acids: ent-lithocholic acid
(ent-LCA),2
ent-chenodeoxycholic acid (ent-CDCA), and
ent-deoxycholic acid (ent-DCA)
(Fig. 1)
(34,
35). Enantiomeric bile acids
have unique farnesoid X receptor, vitamin D receptor, pregnane X receptor, and
TGR5 receptor activation profiles compared with the corresponding natural bile
acids (34). This illustrates
that natural and enantiomeric bile acids interact differently within chiral
environments because of their distinct three-dimensional configurations
(Fig. 1). Despite these
differences in chiral interactions, ent-bile acids have physical
properties identical to those of their natural counterparts including
solubility and critical micelle concentrations
(34,
35). With different receptor
interaction profiles and identical physical properties compared with natural
bile acids, ent-bile acids are ideal compounds to differentiate
between the receptor-mediated and the non-receptor-mediated functions of
natural bile acids.Open in a separate windowFIGURE 1.Natural and enantiomeric bile acids. Structures and
three-dimensional projection views of natural LCA, CDCA, DCA, and their
enantiomers (ent-LCA, ent-CDCA, and ent-DCA). The
three-dimensional ent-steroid structure is rotated 180° around
the long axis for easier comparison with the natural steroid.In this study we explore the enantioselectivity of LCA-, CDCA-, and
DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell
lines, HT-29 and HCT-116. Because the mechanism of natural LCA induced
apoptosis has never been characterized, we then examined in more detail LCA-
and ent-LCA-mediated apoptosis in colon cancer cells. These studies
will not only explore the LCA apoptotic mechanism but will also determine
whether ent-LCA signals through similar cellular pathways. 相似文献
9.
10.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
11.
Harry S. Courtney Yi Li Waleed O. Twal W. Scott Argraves 《The Journal of biological chemistry》2009,284(19):12966-12971
The adhesion of bacteria to host tissues is often mediated by interactions
with extracellular matrices. Herein, we report on the interactions of the
group A streptococcus, Streptococcus pyogenes, with the extracellular
matrix protein fibulin-1. S. pyogenes bound purified fibulin-1 in a
dose-dependent manner. Genetic ablation of serum opacity factor (SOF), a
virulence determinant of S. pyogenes, reduced binding by ∼50%,
and a recombinant peptide of SOF inhibited binding of fibulin-1 to
streptococci by ∼45%. Fibulin-1 bound to purified SOF2 in a dose-dependent
manner with high affinity (Kd = 1.6 nm). The
fibulin-1-binding domain was localized to amino acid residues 457–806 of
SOF2, whereas the fibronectin-binding domain is contained within residues
807–931 of SOF2, indicating that these two domains are separate and
distinct. Fibulin-1 bound to recombinant SOF from M types 2, 4, 28, and 75 of
S. pyogenes, indicating that the fibulin-1-binding domain is likely
conserved among SOF from different serotypes. Mixed binding experiments
suggested that gelatin, fibronectin, fibulin-1, and SOF form a quaternary
molecular complex that enhanced the binding of fibulin-1. These data indicate
that S. pyogenes can interact with fibulin-1 and that SOF is a major
streptococcal receptor for fibulin-1 but not the only receptor. Such
interactions with fibulin-1 may be involved in the adhesion of S.
pyogenes to extracellular matrices of the host.Adhesion of bacteria to host surfaces is the first stage in establishing
bacterial infections in the human host, and a variety of molecular mechanisms
are utilized to initiate adhesion. A common mechanism for adhesion involves
interactions between bacterial adhesins and components of the extracellular
matrices of the host. The identification and characterization of microbial
surface components recognizing adhesive matrix molecules (MSCRAMM) has led to
important advances in vaccines and immunotherapies for preventing and treating
bacterial infections (1).The group A streptococcus, Streptococcus pyogenes, is a major
human pathogen causing diseases ranging from relative minor infections such as
pharyngitis and cellulitis to severe infections with high levels of morbidity
and mortality such as necrotizing fasciitis and toxic shock syndrome
(2). This pathogen expresses
adhesins that interact with various components of the extracellular matrix
including laminin, elastin, fibronectin, fibrinogen, and collagen
(3–7).
The interactions between fibronectin and S. pyogenes have been
intensely studied, and these investigations have revealed at least 10
different streptococcal proteins that bind fibronectin
(4).Serum opacity factor
(SOF)2 is a major
fibronectin-binding protein that is involved in adhesion to host cells
(8–11).
SOF is a virulence determinant that is expressed by approximately half of the
clinical isolates of S. pyogenes
(8). SOF opacifies serum by
binding and displacing apoA-I in high density lipoproteins
(8,
12–15).
SOF is covalently linked to the streptococcal cell wall via an LPSTG sortase
recognition site and is also released in a soluble form. SOF has two
functionally distinct domains, an N-terminal domain that opacifies serum and a
C-terminal domain that binds fibronectin. The role of SOF in adhesion involves
both its C-terminal fibronectin-binding domain and an N-terminal region (see
Fig. 1 for a schematic of
structure) (9,
11). However, the nature of
the interactions between the N-terminal region of SOF and host components is
not well characterized.Open in a separate windowFIGURE 1.A, a schematic of the structure of SOF and its functional domains
is shown. The assignment of functional domains are based on the findings of
Rakonjac et al. (33),
Kreikemeyer et al.
(34), Courtney et al.
(8,
13), and results presented in
this work. Fn, fibronectin. B, the data for the binding of
SOF peptides to fibronectin are from previous publications
(8,
13), and the data for
fibulin-1 are from the present work.Herein, we report on the interactions between a truncated form of SOF in
which its fibronectin-binding domain has been deleted and the extracellular
matrix protein fibulin-1. Fibulin-1 is a member of the fibulin family that
currently consists of seven glycoproteins. All fibulins contain epidermal
growth factor-like repeats and a unique fibulin-type module at its C terminus
that define this family (16,
17). Fibulin-1 is found within
the extracellular matrices and in human plasma at 30–50 μg/ml
(18). It interacts with many
of the components of the extracellular matrix including fibronectin, laminin,
fibrinogen, nidogen-1, endostatin, aggrecan, and versican
(16,
19). Due to its intimate
relationship with the extracellular matrix, it is not surprising that the
defects in fibulin-1 have a wide-ranging impact. Genetic evidence suggests
that fibulin-1 is involved in tissue organization, the maturation and
maintenance of blood vessels, and multiple embryonic pathways
(16,
20–22).Although it has been established that many of the other components of the
extracellular matrix can interact with bacteria, there has been no previous
report on the binding of fibulins to bacteria. Our findings indicate that
fibulin-1 does bind to streptococci and that SOF is a major streptococcal
receptor for fibulin-1. 相似文献
12.
Takafumi Tasaki Adriana Zakrzewska Drew D. Dudgeon Yonghua Jiang John S. Lazo Yong Tae Kwon 《The Journal of biological chemistry》2009,284(3):1884-1895
The N-end rule pathway is a ubiquitin-dependent system where E3 ligases
called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and
type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have
recently reported an E3 family (termed UBR1 through UBR7) characterized by the
70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured
during affinity-based proteomics with synthetic degrons. Here we characterized
substrate binding specificity and recognition domains of UBR proteins.
Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and
300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding
assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations
narrow down the degron-binding activity to a 72-residue UBR box-only fragment
that recognizes type-1 but not type-2 residues. A surface plasmon resonance
assay shows that the UBR box binds to the type-1 substrate Arg-peptide with
Kd of ∼3.4 μm. Downstream from the UBR
box, we identify a second substrate recognition domain, termed the N-domain,
required for type-2 substrate recognition. The ∼80-residue N-domain shows
structural and functional similarity to 106-residue Escherichia coli
ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box
functions as a common structural element essential for binding to all known
destabilizing N-terminal residues, whereas specific residues localized in the
UBR box (for type 1) or the N-domain (for type 2) provide substrate
selectivity through interaction with the side group of an N-terminal amino
acid. Our work provides new insights into substrate recognition in the N-end
rule pathway.The N-end rule pathway is a ubiquitin
(Ub)2-dependent
proteolytic system in which N-terminal residues of short-lived proteins
function as an essential component of degradation signals (degrons) called
N-degrons (Fig. 1A)
(1-15).
An N-degron can be created from a pre-N-degron through specific N-terminal
modifications (12).
Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing
residues that function through their deamidation by N-terminal amidohydrolases
into the secondary destabilizing N-terminal residues Asp and Glu, respectively
(6,
16)
(Fig. 1A). N-terminal
Asp and Glu are secondary destabilizing residues that function through their
arginylation by ATE1 R-transferase, which creates the primary
destabilizing residue Arg at the N terminus
(4,
8)
(Fig. 1A). N-terminal
Cys can also function as a tertiary destabilizing residue through its
oxidation in a manner depending on nitric oxide and oxygen (O2);
the oxidized Cys residue is subsequently arginylated by ATE1
(8,
13,
17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are
indicated by single-letter abbreviations for amino acids. Yellow
ovals denote the rest of a protein substrate. C*
denotes oxidized N-terminal Cys, either Cys-sulfinic acid
[CysO2(H)] or Cys-sulfonic acid [CysO3(H)]. The Cys
oxidation requires nitric oxide and oxygen (O2) or its derivatives.
The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase
(R-transferase). N-recognins also recognize internal (non-N-terminal)
degrons in other substrates of the N-end rule pathway. B, the
X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal
Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue
was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl
beads. Right, the otherwise identical 12-mer peptide, bearing
C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the
streptavidin-Sepharose beads. C, the X-peptide pull-down assay of
endogenous UBR proteins using testes extracts. Extracts from mouse testes were
mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly
(G), or Arg (R). After centrifugation, captured proteins
were separated and subjected to anti-UBR immunoblotting. Mo, a
pull-down reaction with mock beads. D, the X-peptide pull-down assays
using rat testis extracts were performed in the presence of varying
concentrations of NaCl. After incubation and washing, bound proteins were
eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala
for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for
Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and
UBR5. E, cytoplasmic fractions of wild-type (+/+),
Ubr1-/-, Ubr2-/-,
Ubr1-/-Ubr2-/-, and
Ubr1-/-Ubr2-/-Ubr4RNAi
MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were
separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal
residues are directly bound by specific E3 Ub ligases called N-recognins
(3,
7,
9). Destabilizing N-terminal
residues can be created through the removal of N-terminal Met or the
endoproteolytic cleavage of a protein, which exposes a new amino acid at the N
terminus (12,
13). N-terminal degradation
signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2
(bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues
(2,
12). In addition to a
destabilizing N-terminal residue, a functional N-degron requires at least one
internal Lys residue (the site of a poly-Ub chain formation) and a
conformational feature required for optimal ubiquitylation
(1,
2,
18). UBR1 and UBR2 are
functionally overlapping N-recognins
(3,
7,
9). Our proteomic approach
using synthetic peptides bearing destabilizing N-terminal residues captured a
set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa
UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR
box
(10-12).
UBR5 is a HECT E3 ligase known as EDD (E3 identified by
differential display)
(19) and a homolog of
Drosophila hyperplastic discs
(20). The mammalian genome
encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7
(10). UBR box proteins are
generally heterogeneous in size and sequence but contain, with the exception
of UBR4, specific signatures unique to E3s or a substrate recognition subunit
of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in
UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7
(Fig. 2B). The
biochemical properties of more recently identified UBR box proteins, such as
UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and
type-2 destabilizing N-terminal residues. A, the X-peptide
pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in
S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7
(in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for
UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in
B or autoradiography (for UBR6 and UBR7). B, the structures
of UBR box proteins. Shown are locations of the UBR box, the N-domain, and
other E3-related domains. UBR, UBR box; RING, RING finger;
UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich
domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac
development and signaling, angiogenesis
(8,
15), meiosis
(9), DNA repair
(21), neurogenesis
(15), pancreatic functions
(22), learning and memory
(23,
24), female development
(9), muscle atrophy
(25), and olfaction
(11). Mutations in human
UBR1 is a cause of Johanson-Blizzard syndrome
(22), an autosomal recessive
disorder with multiple developmental abnormalities
(26). Other functions of the
pathway include: (i) a nitric oxide and oxygen (O2) sensor
controlling the proteolysis of RGS4, RGS5, and RGS16
(8,
13,
17), (ii) a heme sensor
through hemin-dependent inhibition of ATE1 function
(27), (iii) the regulation of
short peptide import through the peptide-modulated degradation of the
repressor of the import (28,
29), (iv) the control of
chromosome segregation through the degradation of a separate produced cohesin
fragment (30), (v) the
regulation of apoptosis through the degradation of a caspase-processed
inhibitor of apoptosis (31,
32), (vi) the control of the
human immunodeficiency virus replication cycle through the degradation of
human immunodeficiency virus integrase
(10,
33), and (vii) the regulation
of leaf senescence in plants
(34).In the present study we characterized substrate binding specificities and
recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4,
and UBR5 were captured by N-terminal degradation determinants, whereas UBR3,
UBR6, and UBR7 were not. We also report that in contrast to other E3 systems
that usually recognize substrates through protein-protein interface, UBR1 and
UBR2 have a general substrate recognition domain termed the UBR box.
Remarkably, a 72-residue UBR box-only fragment fully retains its structural
integrity and thereby the ability to recognize type-1 N-end rule substrates.
We also report that the N-domain, structurally and functionally related with
bacterial N-recognins, is required for recognizing type-2 N-end rule
substrates. We discuss the evolutionary relationship between eukaryotic and
prokaryotic N-recognins. 相似文献
13.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
14.
Heather B. Claxton David L. Akey Monica K. Silver Suzanne J. Admiraal Janet L. Smith 《The Journal of biological chemistry》2009,284(8):5021-5029
Two thioesterases are commonly found in natural product biosynthetic
clusters, a type I thioesterase that is responsible for removing the final
product from the biosynthetic complex and a type II thioesterase that is
believed to perform housekeeping functions such as removing aberrant units
from carrier domains. We present the crystal structure and the kinetic
analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide
synthetases/polyketide synthase rifamycin biosynthetic cluster of
Amycolatopsis mediterranei. Steady-state kinetics show that RifR has
a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm
of the acyl carrier domain over the hydrolysis of acyl units from the
phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the
decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA
and methylmalonyl-CoA. Multiple RifR conformations and structural similarities
to other thioesterases suggest that movement of a helical lid controls access
of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases
(PKS)3 and
nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed
of modules that work in succession to synthesize secondary metabolites, many
of which are precursors of potent antibiotics, immunosuppressants, anti-tumor
agents, and other bioactive compounds. Rifamycin, the precursor to the
anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line
complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10
PKS modules (1). Each module in
an assembly line complex extends and modifies the intermediate compound before
passing it on to the next module in the series
(Fig. 1A). The
intermediate compounds are covalently attached through a thioester linkage to
the phosphopantetheine arm (Ppant) of carrier domains, one associated with
each module, until they are released from the synthase, usually by a type I
thioesterase (TEI) (2,
3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain
elongation by a PKS module. The chain elongation intermediate is transferred
from the ACP of the upstream module to the ketosynthase (KS) domain.
The acyltransferase (AT) domain transfers an acyl group building
block from CoA to the ACP within the module. The KS domain catalyzes
condensation of the new building block with the intermediate, releasing
CO2. B, production of a decarboxylated acyl unit by the
ketosynthase domain and the subsequent hydrolysis by a TEII. C,
mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a
promiscuous phosphopantetheinyl transferase (Pptase) and the
subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid
derivative by a TEII from an NRPS module comprising an adenylation domain
(A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line
complex and remove the final product through macrocyclization or hydrolysis.
Occasionally, tandem type I thioesterases are integrated at the C terminus of
the final module of NRPS pathways
(4).Although TEIs are covalently attached to the terminal module and generally
process only the final product of an assembly line complex, type II
thioesterases (TEIIs) are discrete proteins that can remove intermediates from
any module in the complex. A variety of functions have been attributed to
TEIIs, the most prevalent of which is a “housekeeping function,”
the removal of aberrant acyl units from carrier domains. These aberrant acyl
units may be due to premature decarboxylation by a PKS ketosynthase domain
(5)
(Fig. 1B) or to
mispriming of the carrier domain by a promiscuous phosphopantetheinyl
transferase
(6–8)
(Fig. 1C). Other
proposed functions for TEIIs include the removal of intermediates from the
synthase as in the case of the mammary gland rat fatty acid synthase (FAS)
TEII in lactating rats, which removes medium chain
C8-C12 fatty acids from the ACP domain
(9) and the removal of amino
acid derivatives from a carrier domain
(10–13),
allowing these derivatives to be incorporated into the natural product by a
later module in the assembly line complex
(Fig. 1D).Disruption of the TEI function results in a complete loss of product,
whereas disruption of TEII function results in a significant decrease in
product yield (30–95%)
(4,
14–24).
Removal of the TEII from the rifamycin assembly line resulted in a 60%
decrease in product yield
(25). Neither TEIs nor TEIIs
may rescue the disrupted function of the other
(6), but a TEII from another
pathway may rescue the function of a disrupted TEII
(26).Two models have been proposed for the TEII housekeeping function
(5). In the high specificity
model, the TEII scans the complex and efficiently removes only aberrant acyl
units. In the low specificity model, the TEII removes both correct and
incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl
units are quickly incorporated into the growing intermediate compound. In
contrast, incorrect acyl units stall the assembly line, providing a longer
window of opportunity for removal by a TEII. Thus a slow, low specificity
enzyme can be effective.TEIIs from different pathways have differing specificities, but general
trends include a preference for decarboxylated acyl units over carboxylated
acyl units (5,
6,
27), substrates linked to a
carrier domain over substrates linked to CoA or the phosphopantetheine mimic
N-acetylcysteamine (7,
28), and single amino acids
over di- or tri-peptides (6,
7). TEIIs are able to hydrolyze
substrates attached to carrier domains from their native pathway as well as
other pathways (6,
20,
28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family.
Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal
structures for the TEIs from the pikromycin (PikTE) PKS
(29), 6-deoxyerythronolide B
(DEBSTE) PKS (30), surfactin
NRPS (SrfTE) (31), fengycin
NRPS (FenTE) (32), and human
fatty acid synthase (hFasTE)
(33) systems, and NMR
structures for enterobactin TEI
(34), and surfactin TEII
(35). Like PKS modules, PKS
TEIs are dimers. The dimer interface comprises two N-terminal helices that are
unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS
TEII of surfactin is also monomeric
(31). Although the FAS complex
is dimeric, the FAS TEI is a monomer
(33). All of the TEs have an
α-helical insertion after strand β5 that forms a lid over the
active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming
helices contribute to the lid structure, forming a fixed channel that runs the
length of the TE and contains the active site. In contrast, the active site
pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the
lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal
structure (7), and chemical
shift observations suggested greater flexibility for residues of the lid
region in the surfactin TEII (SrfTEII) solution structure
(35). These movements seem to
be of functional importance, because a movement of a linker peptide in SrfTEI
determines the shape of the active site pocket and a movement of the first lid
helix appears to modulate access to the active site
(31).We report the structure and activity of recombinant RifR, the TEII of the
rifamycin biosynthetic cluster. Steady-state kinetic analysis of the
hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP
substrates demonstrates that acyl-ACP substrates are preferred over the
acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently
than are the natural rifamycin building blocks. We report the crystal
structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape
of the substrate chamber are variable, because one of the elements forming the
chamber, an extended linker segment, is highly flexible, and different crystal
forms reveal different shapes for the substrate binding site. Access to the
active site is severely restricted, and structural comparisons with other
thioesterases suggest that a conformational change in the lid and the flexible
linker region is required for access to the substrate pocket. 相似文献
15.
16.
Tsuneo Ferguson Jitesh A. Soares Tanja Lienard Gerhard Gottschalk Joseph A. Krzycki 《The Journal of biological chemistry》2009,284(4):2285-2295
Archaeal methane formation from methylamines is initiated by distinct
methyltransferases with specificity for monomethylamine, dimethylamine, or
trimethylamine. Each methylamine methyltransferase methylates a cognate
corrinoid protein, which is subsequently demethylated by a second
methyltransferase to form methyl-coenzyme M, the direct methane precursor.
Methylation of the corrinoid protein requires reduction of the central cobalt
to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric
iron-sulfur protein, was isolated from Methanosarcina barkeri and is
required for in vitro ATP-dependent reductive activation of
methylamine:CoM methyl transfer from all three methylamines. In the absence of
the methyltransferases, highly purified RamA was shown to mediate the
ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for
the monomethylamine corrinoid protein, MtmC. The ramA gene is located
near a cluster of genes required for monomethylamine methyltransferase
activity, including MtbA, the methylamine-specific CoM methylase and the
pyl operon required for co-translational insertion of pyrrolysine
into the active site of methylamine methyltransferases. RamA possesses a
C-terminal ferredoxin-like domain capable of binding two tetranuclear
iron-sulfur proteins. Mutliple ramA homologs were identified in
genomes of methanogenic Archaea, often encoded near methyltrophic
methyltransferase genes. RamA homologs are also encoded in a diverse selection
of bacterial genomes, often located near genes for corrinoid-dependent
methyltransferases. These results suggest that RamA mediates reductive
activation of corrinoid proteins and that it is the first functional archetype
of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon
dioxide. The Methanosarcinaceae are a notable exception as representatives are
capable of methylotrophic methanogenesis from methylated amines, methylated
thiols, or methanol. Methanogenesis from these substrates requires methylation
of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used
by methylreductase to generate methane and a mixed disulfide whose reduction
leads to energy conservation
(1–4).Methylation of CoM with trimethylamine
(TMA),4 dimethylamine
(DMA), or monomethylamine (MMA) is initiated by three distinct
methyltransferases that methylate cognate corrinoid-binding proteins
(3). MtmB, the MMA
methyltransferase, specifically methylates cognate corrinoid protein, MtmC,
with MMA (see Fig. 1)
(5,
6). The DMA methyltransferase,
MtbB, and its cognate corrinoid protein, MtbC, interact specifically to
demethylate DMA (7,
8). TMA is demethylated by the
TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein
(MttC) (8,
9). Each of the methylated
corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase,
MtbA, which produces methyl-CoM
(10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by
MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the
catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the
supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl
transfer from MMA to the thiolate of coenzyme M (HSCoM). In
vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to
the neutral species or with RamA in an ATP-dependent reaction can regenerate
the Co(I) species. In either case in vitro Ti(III)-citrate is the
ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the
corrinoid protein MtaC, which is then demethylated by another
methylcobamide:CoM methyltransferase, MtaA
(13–15).
The methylation of CoM with methylated thiols such as dimethyl sulfide in
Methanosarcina barkeri is catalyzed by a corrinoid protein that is
methylated by dimethyl sulfide and demethylated by CoM, but in this case an
associated CoM methylase carries out both methylation reactions
(16).In bacteria, analogous methyltransferase systems relying on small corrinoid
proteins are used to achieve methylation of tetrahydrofolate. In
Methylobacterium spp., CmuA, a single methyltransferase with a
corrinoid binding domain, along with a separate pterin methylase, effect the
methylation of tetrahydrofolate with chloromethane
(17,
18). In Acetobacterium
dehalogenans and Moorella thermoacetica various three-component
systems exist for specific demethylation of different phenylmethyl ethers,
such as vanillate (19) and
veratrol (20), again for the
methylation of tetrahydrofolate. Sequencing of the genes encoding the
corrinoid proteins central to the archaeal and bacterial methylotrophic
pathways revealed they are close homologs. Furthermore, genes predicted to
encode such corrinoid proteins and pterin methyltransferases are widespread in
bacterial genomes, often without demonstrated metabolic function. All of these
corrinoid proteins are similar to the well characterized cobalamin binding
domain of methionine synthase
(21,
22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not
homologous proteins. The methylamine methyltransferases do share the common
distinction of having in-frame amber codons
(6,
8) within their encoding genes
that corresponds to the genetically encoded amino acid pyrrolysine
(23–25).
Pyrrolysine has been proposed to act in presenting a methylammonium adduct to
the central cobalt ion of the corrinoid protein for methyl transfer
(3,
23,
26). However, nucleophilic
attack on a methyl donor requires the central cobalt ion of a corrinoid
cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II)
state (27). Subsequent
demethylation of the methyl-Co(III) corrinoid cofactor regenerates the
nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain
of methionine synthase has an Em value of -525 mV at pH 7.5
(28). It is likely to be
similarly low in the homologous methyltrophic corrinoid proteins. These low
redox potentials make the corrinoid cofactor subject to adventitious oxidation
to the inactive Co(II) state (Fig.
1).During isolation, these corrinoid proteins are usually recovered in a
mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies,
chemical reduction can maintain the corrinoid protein in the active Co(I)
form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate
methyltransferase systems can be activated in vitro by the addition
of Ti(III) alone as an artificial reductant
(14,
19). In contrast, activation
of the methylamine corrinoid proteins further requires the addition of methyl
viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely
low potential neutral species. In vitro activation with these agents
does not require ATP (5,
7,
9).Cellular mechanisms also exist to achieve the reductive activation of
corrinoid cofactors in methyltransferase systems. Activation of human
methionine synthase involves reduction of the co(II)balamin by methionine
synthase reductase (29),
whereas the Escherichia coli enzyme requires flavodoxin
(30). The endergonic reduction
is coupled with the exergonic methylation of the corrinoid with
S-adenosylmethionine
(27). An activation system
exists in cellular extracts of A. dehalogenans that can activate the
veratrol:tetrahydrofolate three-component system and catalyze the direct
reduction of the veratrol-specific corrinoid protein to the Co(I) state;
however, the activating protein has not been purified
(31).For the methanogen methylamine and methanol methyltransferase systems, an
activation process is readily detectable in cell extracts that is ATP- and
hydrogen-dependent (32,
33). Daas et al.
(34,
35) examined the activation of
the methanol methyltransferase system in M. barkeri and purified in
low yield a methyltransferase activation protein (MAP) which in the presence
of a preparation of hydrogenase and uncharacterized proteins was required for
ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was
found to be a heterodimeric protein without a UV-visible detectable prosthetic
group. Unfortunately, no protein sequence has been reported for MAP, leaving
the identity of the gene in question. The same MAP protein was also suggested
to activate methylamine:CoM methyl transfer, but this suggestion was based on
results with crude protein fractions containing many cellular proteins other
than MAP (36).Here we report of the identification and purification to near-homogeneity
of RamA (reductive activation of
methyltransfer, amines), a protein mediating activation
of methylamine:CoM methyl transfer in a highly purified system
(Fig. 1). Quite unlike MAP,
which was reported to lack prosthetic groups, RamA is an iron-sulfur protein
that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I)
state in an ATP-dependent reaction (Fig.
1). Peptide mapping of RamA allowed identification of the gene
encoding RamA and its homologs in the genomes of Methanosarcina spp.
RamA belongs to COG3894, a group of uncharacterized metal-binding proteins
found in a number of genomes. RamA, thus, provides a functional example for a
family of proteins widespread among bacteria and Archaea whose physiological
role had been largely unknown. 相似文献
17.
Akiko Maeda Tadao Maeda Marcin Golczak Steven Chou Amar Desai Charles L. Hoppel Shigemi Matsuyama Krzysztof Palczewski 《The Journal of biological chemistry》2009,284(22):15173-15183
Exposure to bright light can cause visual dysfunction and retinal
photoreceptor damage in humans and experimental animals, but the mechanism(s)
remain unclear. We investigated whether the retinoid cycle (i.e. the
series of biochemical reactions required for vision through continuous
generation of 11-cis-retinal and clearance of
all-trans-retinal, respectively) might be involved. Previously, we
reported that mice lacking two enzymes responsible for clearing
all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding
cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal
abnormalities exacerbated by light and associated with accumulation of
diretinoid-pyridinium-ethanolamine (A2E), a condensation product of
all-trans-retinal and a surrogate marker for toxic retinoids. Now we
show that these mice develop an acute, light-induced retinopathy. However,
cross-breeding these animals with lecithin:retinol acyltransferase knock-out
mice lacking retinoids within the eye produced progeny that did not exhibit
such light-induced retinopathy until gavaged with the artificial chromophore,
9-cis-retinal. No significant ocular accumulation of A2E occurred
under these conditions. These results indicate that this acute light-induced
retinopathy requires the presence of free all-trans-retinal and not,
as generally believed, A2E or other retinoid condensation products. Evidence
is presented that the mechanism of toxicity may include plasma membrane
permeability and mitochondrial poisoning that lead to caspase activation and
mitochondria-associated cell death. These findings further understanding of
the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate
retina responsible for continuous generation of 11-cis-retinal from
its all-trans-isomer
(1-3).
Because 11-cis-retinal is the chromophore of rhodopsin and cone
visual pigments (4), disabling
mutations in genes encoding proteins of the retinoid cycle can cause a
spectrum of retinal diseases affecting sight
(3). Moreover, the efficiency
of the mammalian visual system and health of photoreceptors and retinal
pigment epithelium
(RPE)2 decrease
significantly with age. Even in the presence of a functional retinoid cycle,
A2E, retinal dimer (RALdi), and other toxic all-trans-retinal
condensation products
(5-7)
can accumulate as a consequence of aging
(8). Under experimental
conditions, these compounds can produce toxic effects on RPE cells
(9-11).
Patients affected by age-related macular degeneration, Stargardt disease, or
other retinal diseases associated with accumulation of surrogate markers, such
as A2E, all develop retinal degeneration
(12). Thus, elucidating the
fundamental causes of these age-dependent changes is of increasing importance.
Encouragingly, our understanding of both retinoid metabolism outside the eye
and production of 11-cis-retinal unique to the eye has accelerated
recently (Scheme 1)
(1-3),
and genetic mouse models are readily available to study these processes and
their potential aberrations in vivo
(13). Thus, a central question
can be addressed, namely what initiates the death of photoreceptor cells and
the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual
cycle. After diffusion from the RPE, the visual chromophore,
11-cis-retinal, combines with rhodopsin and then is photoisomerized
to all-trans-retinal. Most of the all-trans-retinal
dissociates from opsin into the cytoplasm, where it is reduced to
all-trans-retinol by RDHs, including RDH8. The fraction of
all-trans-retinal that dissociates into the disc lumen is transported
by ABCA4 into the cytoplasm
(23) before it is reduced.
All-trans-retinol then is translocated to the RPE, esterified by
LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are
associated with human macular degeneration, Stargardt disease, and age-related
macular degeneration (55,
56).Several mechanisms associated with retinoid metabolism may contribute to
different retinopathies (1).
For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or
chromophore in retinoid isomerase knock-out (Rpe65-/-)
mice leads to rapid degeneration of cone photoreceptors and slowly progressive
death of rods (14). Such mice
do not produce toxic condensation products from all-trans-retinal.
Instead, their retinopathies have been attributed to continuous activation of
visual phototransduction (15)
due to either the basal activity of opsin
(16-18)
or disordered vectorial transport of cone visual pigments without bound
chromophore (19).
Paradoxically, an abnormally high flux of retinoids through the retinoid cycle
can also lead to retinopathy in other mouse models
(20,
21). Animal models featuring
anomalies in the retinoid cycle illustrate the importance of chromophore
regeneration and provide an approach to elucidating mechanisms involved in
human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8
(retinol dehydrogenase 8), one of the main enzymes that reduces
all-trans-retinal in rod and cone outer segments
(22), and Abca4
(ATP-binding cassette transporter 4), which transports
all-trans-retinal from the inside to the outside of disc membranes
(23), rapidly accumulate
all-trans-retinal condensation products and exhibit accentuated
RPE/photoreceptor dystrophy at an early age
(24). Although these studies
suggest retinoid toxicity, it is still unclear if the elevated levels of
retinal and/or its condensation products, such as A2E, are the cause of this
retinopathy or merely a nonspecific reflection of impaired retinoid
metabolism. Here, we report that spent chromophore,
all-trans-retinal, is most likely responsible for photoreceptor
degeneration in Rdh8-/-Abca4-/- mice.
Toxic effects of all-trans-retinal include caspase activation and
mitochondria-associated cell death. 相似文献
18.
19.
Neil Portman Sylvain Lacomble Benjamin Thomas Paul G. McKean Keith Gull 《The Journal of biological chemistry》2009,284(9):5610-5619
Eukaryotic flagella from organisms such as Trypanosoma brucei can
be isolated and their protein components identified by mass spectrometry. Here
we used a comparative approach utilizing two-dimensional difference gel
electrophoresis and isobaric tags for relative and absolute quantitation to
reveal protein components of flagellar structures via ablation by inducible
RNA interference mutation. By this approach we identified 20 novel components
of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of
these as being present within the PFR by immunofluorescence. Bioinformatic
analysis of the PFR cohort reveals a likely calcium/calmodulin
regulatory/signaling linkage between some components. We extended the RNA
interference mutant/comparative proteomic analysis to individual novel
components of our PFR proteome, showing that the approach has the power to
reveal dependences between subgroups within the cohort.The eukaryotic cilium/flagellum is a multifunctional organelle involved in
an array of biological processes ranging from cell motility to cell signaling.
Many cells in the human body, across a range of tissues and organs, produce
either single or multiple, motile or nonmotile cilia where they perform
diverse biological processes essential for maintaining human health. This
diversity of function is reflected in an equally diverse range of pathologies
and syndromes that result from ciliary/flagellar dysfunction via inherited
mutations. This diversity is a reflection of the molecular complexity, both in
components and in protein interactions of this organelle
(1,
2).The canonical eukaryotic flagellum displays a characteristic “9 +
2” microtubular profile, where nine outer doublet microtubules encircle
two singlet central pair microtubules, an arrangement found in organisms as
diverse as trypanosomes, green algae, and mammals. Although this 9 + 2
microtubule arrangement has been highly conserved through eukaryotic
evolution, there are examples where this standard layout has been modified,
including the “9 + 0” layout of primary cilia and the “9 + 9
+ 2” of many insect sperm flagella. In addition to this highly conserved
9 + 2 microtubule structure, flagella and cilia show a vast range of discrete
substructures, such as the inner and outer dynein arms, nexin links, radial
spokes, bipartite bridges, beak-like projections, ponticuli, and other
microtubule elaborations that are essential for cilium/flagellum function.
Cilia and flagella can also exhibit various extra-axonemal elaborations, and
although these are often restricted to specific lineages, there is evidence
that some functions, such as metabolic specialization, provided by these
diverse structures are conserved
(3,
4). Examples of such
extraaxonemal elaborations include the fibrous or rod-like structures in the
flagellum of the parasite Giardia lamblia
(5), kinetoplastid protozoa
(6,
7), and mammalian sperm
flagella, along with extra sheaths of microtubules in insect sperm flagella
(8).Several recent studies have set out to determine the protein composition of
the flagellum and demonstrated the existence of both an evolutionarily
conserved core of flagellum/cilium proteins and a large number of
lineage-restricted components
(9–13).
Although these approaches provide an invaluable catalogue of the protein
components of the flagellum, they provide only limited information on the
substructural localization of proteins and do not address either the likely
protein-protein interactions or the function of these proteins within the
flagellum. To address these issues, the protein composition of some axonemal
substructures (radial spoke complexes; for example see Ref.
14) has been determined by
direct isolation of these structures, and a number of complexes have been
resolved by the use of co-immunoprecipitation of indicator proteins (for
example see Refs. 15 and
16). In addition the
localization and function of a number of flagellar proteins have been
investigated by detailed analysis of mutant cell lines (particularly of
Chlamydomonas reinhardtii) that exhibit defined structural defects
within the assembled axoneme. Early studies employed two-dimensional PAGE to
compare the proteomic profile of purified flagella derived from C.
reinhardtii mutants and wild type cells
(17–22)
that showed numerous proteomic differences in the derived profiles. The
available technology did not allow identification of the individual proteins
within the profiles. Recent proteomic advances offer the opportunity for this
identification. For instance the comparative proteomic technique isotope coded
affinity tagging has been used to identify components of the outer dynein arm
(23). This technique utilizes
stable isotope tagging to quantify the relative concentration of proteins
between two samples.Trypanosomatids are important protozoan parasites whose flagellum is a
critical organelle for their cell biology and pathogenicity. Their
experimental tractability also provides opportunities for generic insights to
the eukaryotic flagellum. They are responsible for a number of devastating
diseases of humans and other mammals, including commercially important
livestock, in some of the poorest areas of the world
(24–26).
All kinetoplastids build a flagellum that contains an extra-axonemal structure
termed the paraflagellar rod
(PFR).3 In the case of
the African trypanosome Trypanosoma brucei brucei, this consists of a
complex subdomain organization of a proximal, intermediate, and distal domain
as well as links to specific doublets of the axoneme and a structure known as
the flagellum attachment zone (FAZ) by which the flagellum is attached to the
cell body for much of its length
(6,
7). The PFR is required for
cell motility (27,
28) and serves as a scaffold
for metabolic and signaling enzymes
(3,
29,
30). We have previously shown
that the presence of this structure is essential for the survival of the
mammalian bloodstream form of the parasite both in vitro (in culture)
(12) and in vivo (in
mice) (31) as part of a wider
requirement for motility in this life cycle stage
(12,
32,
33).Two major protein components of the PFR (PFR1 and PFR2) have been
identified
(34–38)
along with several minor PFR protein components
(3,
29,
30,
39–43).
The availability of RNAi techniques in T. brucei allowed the
generation of the inducible mutant cell line snl2
(44), in which RNAi-mediated
ablation of the PFR2 protein causes the specific loss of both the distal and
intermediate PFR subdomains (see Fig.
1A). After RNAi induction cells become paralyzed but
remain viable (44). Our
laboratory (3) has previously
identified two PFR-specific adenylate kinases by comparing two-dimensional
SDS-PAGE gels of purified flagella from induced and noninduced snl2
cells. These proteins cannot be incorporated into the PFR after PFR2
ablation.Open in a separate windowFIGURE 1.A, electron microscopy images (prepared as described previously
(12)) of T. brucei
snl2 noninduced and RNAi-induced flagellar transverse sections shows the
loss of a large part of the PFR structure. Bar, 100 nm. B,
frequencies (resolution 0.25) of log2 protein abundance ratios of
noninduced to noninduced samples from quadruplex iTRAQ. C, averaged
frequencies (resolution 0.25) of log2 protein abundance ratios of
induced to noninduced samples from quadruplex iTRAQ. D,
log2 protein abundance ratios of induced to noninduced samples from
all iTRAQ experiments for all proteins that show at least a 2-fold decrease
after RNAi induction of snl2. α- and β-tubulin show a less
than 2-fold change as expected. The results of individual sample pairs are
graphed separately as per key.The ability to ablate PFR2 and hence disable assembly of a major portion of
the PFR affords an opportunity to apply advanced proteomic approaches to
identify additional PFR proteins. In this present study we have used two
complementary proteomic approaches, two-dimensional fluorescence difference
gel electrophoresis (DIGE)
(45) and isobaric tags for
relative and absolute quantitation (iTRAQ; Applied Biosystems), to investigate
PFR+ and PFR–flagella to define 30 components of these two PFR
subdomains. We have also conducted a bioinformatic analysis of amino acid
motifs present in this protein cohort to gain insights into the possible
functions of novel proteins and used epitope tagging approaches to confirm the
PFR localization of a test set of identified proteins. We then asked whether
it was possible to combine comparative proteomics with further analysis of
RNAi mutant trypanosomes to provide detailed information on the individual
interactions and assembly dependences within the novel PFR components we had
identified. By iterating the subtractive proteomic analysis with novel
putative PFR proteins, we were able to reveal the existence of distinct PFR
protein dependence relationships and provide intriguing new insight into
regulatory processes potentially operating within the trypanosome flagellum.
Finally, this study establishes the mutant/proteomic combination as a powerful
enabling approach for revealing dependences within subcohorts of the flagellar
proteome. 相似文献
20.
Wenli Li Jianhua Ju Scott R. Rajski Hiroyuki Osada Ben Shen 《The Journal of biological chemistry》2008,283(42):28607-28617
Tautomycin (TTM) is a highly potent and specific protein phosphatase
inhibitor isolated from Streptomyces spiroverticillatus. The
biological activity of TTM makes it an important lead for drug discovery,
whereas its spiroketal-containing polyketide chain and rare dialkylmaleic
anhydride moiety draw attention to novel biosynthetic chemistries responsible
for its production. To elucidate the biosynthetic machinery associated with
these novel molecular features, the ttm biosynthetic gene cluster
from S. spiroverticillatus was isolated and characterized, and its
involvement in TTM biosynthesis was confirmed by gene inactivation and
complementation experiments. The ttm cluster was localized to a 86-kb
DNA region, consisting of 20 open reading frames that encode three modular
type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five
proteins for methoxymalonyl-S-acyl carrier protein biosynthesis
(Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and
regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF
and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is
proposed based on functional assignments from sequence analysis, which agrees
well with previous feeding experiments, and has been further supported by
in vivo gene inactivation experiments. These findings set the stage
to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM
analogs.Tautomycin (TTM)2
is a polyketide natural product first isolated in 1987 from Streptomyces
spiroverticillatus (1).
The structure and stereochemistry of TTM were established on the basis of
chemical degradation and spectroscopic evidence
(2-4).
TTM contains several features not common to polyketide natural products,
including a spiroketal group, a methoxymalonate-derived unit, and an acyl
chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is
tautomycetin (TTN), which was first isolated in 1989 from Streptomyces
griseochromogenes following the discovery of TTM
(5,
6). The structure of TTN was
deduced by chemical degradation and spectroscopic analysis
(6), and its stereochemistry
was established by comparison of spectral data with those of TTN degradation
products and synthetic fragments
(7). Both TTM and TTN exist as
tautomeric mixtures composed of two interconverting anhydride and diacid forms
in approximately a 5:4 ratio under neutral conditions
(Fig. 1A)
(1,
2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and
biosynthetic origin of the dialkylmaleic anhydride by feeding experiments
using 13C-labeled acetate and propionate. The
methoxymalonate-derived unit in TTM is highlighted by the dotted oval.
R, polyketide moiety of TTM or TTN. B, selected natural product
inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric
anhydride moiety. C, selected natural products containing a
dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes
in leukemia cells (8). However,
it was later realized that TTM is a potent and specific inhibitor of protein
phosphatases (PPs) PP-1 and PP-2A
(9). PP-1 and PP-2A are two of
the four major serine/threonine protein phosphatases that regulate diverse
cellular events such as cell division, gene expression, muscle contraction,
glycogen metabolism, and neuronal signaling in eukaryotic cells
(10-12).
Many natural product PP-1 and PP-2A inhibitors are known, including okadaic
acid (13), calyculin-A
(14), phoslactomycin,
spirastrellolide, and cantharidin
(15)
(Fig. 1B), as well as
TTM (16,
17), and TTN
(18). They have served as
useful tools to study PP-involved intracellular events in vivo and as
novel leads for drug discovery
(10-12).
Among these PP inhibitors, TTM and TTN are unique because of their PP-1
selectivity. Despite their structural similarities, TTM exhibits potent
specific inhibition of PP-1 and PP-2A with IC50 values of 22-32
nm and only a slight preference for PP-1
(18). Conversely, TTN shows
nearly a 40-fold higher binding affinity to PP-1 (IC50 = 1.6
nm) than to PP-2A (IC50 = 62 nm)
(18). Because the major
structural differences between TTM and TTN reside in the region distal to the
dialkylmaleic anhydride moiety (Fig.
1A), it has been proposed that differences in these
moieties might be responsible for the PP-1 selectivity
(17-19).
Finally, TTN also has an impressive immuno-suppressive activity
(20,
21), which is apparently
devoid for TTM. Clearly, the structural differences between these two
polyketides translate into large, exploitable differences in bio-activities,
yet an understanding of the biosynthetic origins of these differences remains
elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for
polyketide natural products, as is the methoxymalonate-derived unit
(Fig. 1A). Few studies
have been carried out for spiroketal biosynthesis, yet it is reasonably common
among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few
others (Fig. 1B). Less
common, but still found in the phosphatase inhibitor cantharidin, as well as
TTM and TTN, is the dialkylmaleic anhydride moiety
(Fig. 1B); this unit
appears in a number of other natural products
(Fig. 1C), although
the biosynthetic steps leading to this reactive moiety (a protected version of
a dicarboxylate) have not been rigorously investigated. Feeding experiments
with 13C-labeled precursors indicated that the anhydride of TTM and
TTN is assembled from a propionate and an as yet undefined C-5 unit
(Fig. 1A), which would
require novel chemistry for polyketide biosynthesis
(22). TTM differentiates
itself from all known PP-1 and PP-2A inhibitors by virtue of its unique
combination of both the dialkymaleic anhydride and spiroketal
functionalities.Multiple total syntheses of TTM and a small number of analogs have been
reported, confirming the predicted structure and absolute stereochemistry and
facilitating structure-activity relationship studies on PP inhibition and
apoptosis induction (19,
23-25).
These studies revealed that: (i) the C22-C26 carbon chain and the
dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii)
the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition;
(iii) the spiroketal moiety determines the affinity to specific protein
phosphatases; (iv) the active form is most likely the dicarboxylate; and (v)
3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as
a whole, none of the analogs had an improved potency or selectivity for PP-1
inhibition than the natural TTM
(19,
22-25).
As a result, a more specific inhibitor of PP-1 is urgently awaited to
differentiate the physiological roles of PP-1 and PP-2A in vivo and
to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic
gene cluster from S. spiroverticillatus as the first step toward
engineering TTM biosynthesis for novel analogs
(26). We report here: (i)
cloning and sequencing of the complete ttm gene cluster, (ii)
determination of the ttm gene cluster boundaries, (iii)
bioinformatics analysis of the ttm cluster and a proposal for TTM
biosynthesis, and (iv) genetic characterization of the TTM pathway to support
the proposed pathway. Of particular interest has been the identification of
genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling
of the ttm polyketide synthase (PKS) genes predicted to select and
incorporate four different starter and extender units for TTM production, and
the apparent lack of candidate genes associated with spiroketal formation.
These findings now set the stage to engineer TTM analogs for novel PP-1- and
PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to
the TTM biosynthetic machinery. 相似文献