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1.
STUDIES ON THE BIOSYNTHESIS OF COLLAGEN : I. THE GROWTH OF FOWL OSTEOBLASTS AND THE FORMATION OF COLLAGEN IN TISSUE CULTURE 总被引:5,自引:2,他引:5 下载免费PDF全文
1. A tissue culture method was devised in which suspensions of osteoblasts, obtained directly from frontal bones of fowl embryos, were grown in a fluid, fibrin-free medium. 2. Maximum growth of the tissue, as measured by dry weight, with the formation of collagen protein, based on the estimation of hydroxyproline, was obtained in periods of up to 6 days. 3. Appreciable amounts of protein-bound hydroxyproline were formed during the first 24 hour growth period, but electron microscopy of portions of the same cultures failed to demonstrate the presence of any typical collagen fibrils. 4. The subsequent formation of many characteristic collagen fibrils was not associated with a significant rise in the mean hydroxyproline content of the tissue. 5. The cytoplasmic granules of the osteoblasts stained intensely with the P.A.S. technique when the collagen fibrils were being formed. 6. It is suggested that collagen-forming cells synthesise and secrete a hydroxyproline-rich precursor of protein or large peptide nature, which subsequently becomes directly transformed into typical collagen fibrils. 相似文献
2.
Suniti Misra Vincent C. Hascall Carla De Giovanni Roger R. Markwald Shibnath Ghatak 《The Journal of biological chemistry》2009,284(18):12432-12446
Our studies have shown that constitutive interactions between hyaluronan
and CD44 on tumor cells induces various anti-apoptotic cell survival pathways
through the formation of a multimeric signaling complex that contains
activated receptor tyrosine kinases. Inhibition of the hyaluronan-CD44
interactions on tumor cells by hyaluronan-CD44 interaction antagonists
suppresses these activities by disassembling the complex. Although the
anti-tumor activity of hyaluronan-oligosaccharides, a hyaluronan-CD44
interaction antagonist, is effective in sensitizing tumor cells to
chemotherapeutic agents and reducing tumor growth in xenografts,
hyaluronan-oligosaccharide alone was not effective in reducing tumor
progression in Apc Min/+ mice. We now show in vitro and
in vivo that targeted inhibition of the expression of CD44v6 depletes
the ability of the colon tumor cells to signal through hyaluronan-CD44v6
interactions. First, we cloned oligonucleotides coding CD44v6 shRNA into a
conditionally silenced pSico vector. Second, using pSico-CD44v6 shRNA and a
colon-specific Fabpl promoter-driven Cre recombinase expression
vector packaged into transferrin-coated nanoparticles, we successfully
delivered the CD44v6 shRNA within pre-neoplastic and neoplastic colon
malignant cells. Third, using the Apc Min/+ mice model, we
demonstrated that inhibition of the CD44v6 expression reduces the signaling
through a hyaluronan/CD44v6-pErbB2-Cox-2 interaction pathway and
reduced adenoma number and growth. Together, these data provide insight into
the novel therapeutic strategies of short hairpin RNA/nanoparticle technology
and its potential for silencing genes associated with colon tumor cells.Extracellular matrix has a significant role in solid tumor growth
(1). Hyaluronan
(HA)3 is
one of the constituents of extracellular matrix. HA is a high molecular weight
glycosaminoglycan present in almost every tissue of vertebrates. It is
concentrated in regions of high cell division and invasion. Like numerous
extracellular matrix constituents, HA serves both structural and instructive
roles in terms of cell signaling via HA receptors, mainly CD44, on the surface
of most cells
(2-4).
However, when cells proliferate or migrate, e.g. in embryonic
processes, tissue remodeling, inflammation, and diseases such as cancer and
atherosclerosis, HA-induced signaling is activated
(4-6).
High levels of HA in tumors are a prognostic factor in several malignancies
(7), and manipulations of HA
production or interaction with cell surface receptors strongly influence tumor
growth and metastasis (4,
6).CD44 also has an important role in tumor progression
(8). CD44 proteins exist in
three states with respect to HA binding as follows: non-HA binding; nonbinding
unless activated by physiological stimuli; or constitutively activated
(9). Thus HA induces
intracellular signaling when it binds to “constitutively
activated” CD44 variants during cell dynamic processes, but it does not
do so under conditions of adult tissue homeostasis
(10,
11). The CD44 structure of
normal cells is distinct from that of cancer cells, because under various
physiological and pathological conditions, the local environmental pressure
influences alternate splicing and post-translational modification to produce
diversified CD44 molecules (8,
12). This diversification
allows the production of specific targeting agents that will be useful for
both diagnosis and therapy. Overexpression of the variant high molecular
weight isoforms CD44v4-v7 and CD44v6-v9 in human lymphomas, colorectal
adenocarcinomas, endometrial cancer, papillary thyroid carcinoma, lung and
breast cancer, and metastasizing rat adenocarcinomas
(13-20),
as well as down-regulation of standard CD44 (CD44s), are postulated to result
in increased tumorigenicity
(21). Moreover, the systemic
application of antibodies directed against the v6 epitope and the expression
of antisense CD44v6 can retard tumor growth and block metastasis in
vivo (13,
14), emphasizing the potential
importance of CD44 variants as therapeutic targets in cancer, particularly
colon cancer. Indeed overexpression of CD44 is an early event in the
colorectal adenoma-carcinoma sequence
(22-24).
Thus, unlike the targeting of various cancer-promoting molecules, CD44
targeting provides a strong opportunity for discerning therapy in colon cancer
cells.Altered COX-2 gene expression occurs in human colon carcinomas,
and Cox-2 antagonists inhibit colon cancer progression in animal
models (25,
26). A recent study implicates
COX-2 overexpression as a proximal mediator of CD44-dependent
invasion in human non-small cell lung cancer and in human renal carcinoma
cells (27,
28). Cox-2 and CD44v
(23,
24,
29) are overexpressed in
carcinogen-induced tumors, and our recent study demonstrates that HA-CD44
interactions constitutively regulate COX-2-induced cell survival in
normal epithelial cells and colon carcinoma cells
(30,
31).Our previous studies demonstrated that antagonists of HA-CD44 interactions,
i.e. HA oligomers and overexpression of the ectodomain of CD44
(soluble CD44) (32) that acts
as a competitive decoy by binding to endogenous HA, inhibit cell survival
pathway activities, including activation of several receptor tyrosine kinases,
namely ERBB2, EGFR, IGF1Rβ, c-MET, and PDGFRβ, in several types of
malignant colon, breast, and prostate carcinoma cells
(33,
34). In our recent study we
demonstrated that elevated HA in normal intestinal epithelial cells
(HIEC6-HAS2) regulates several properties required for the
transformed phenotype. Increased HA in these cells regulates expression and
enzymatic activity of COX-2, activation of ErbB2 and AKT, and
translocates β-catenin to the nucleus
(30,
31). To explore the mechanism
of constitutive HA-CD44 interaction and the consequent outcomes in cancer
cells, we have shown that all four types of reagents, namely
HA-oligosaccharides, anti-CD44 antibody, soluble CD44, or CD44 siRNA, block
signaling responses in a variety of tumor cell types and also block activation
of receptor tyrosine kinases
(30,
31,
33,
34). Although the anti-tumor
activity of HA-oligosaccharides is effective in sensitizing tumor cells to
chemotherapeutic agents in cell culture models
(34-36)
and in reducing tumor growth in a subcutaneous in vivo xenograft
model (37),
HA-oligosaccharides alone are not effective in reducing tumor progression in
an Apc Min/+ mice in vivo
model.4
Thus, an alternative HA/CD44 antagonist is required to treat distant tumors.
Because CD44 is present in the HA-induced signaling complex, we also used RNA
interference to test the role of CD44 in these cancer cells
(33,
34). All cancer cell lines
(HCT 116 and HCA7 colon cancer cells, C4-2 and LNCaP prostate cancer cells,
and MCF7/Adr breast cancer cells and TA3St mouse mammary carcinoma cells)
transfected with a 21-nucleotide small interfering RNA (siRNA) targeted to
CD44 greatly decreased activation of cell survival proteins and multiple
receptor tyrosine kinases as well as CD44 and COX-2 expression when
compared with the above cells transfected with scrambled control siRNA
(30,
31,
34).In mammalian cell culture, the transfection of 21-nucleotide
double-stranded siRNA efficiently inhibits endogenous gene expression in a
sequence-specific manner without induction of the interferon response
(38). However, the phenotypic
changes induced by siRNAs only persist 1 week because of lack of transfer of
siRNA or dilution of siRNA concentration after each cell division, which
limits their utility for use in inhibiting tumor progression. The technique of
using shRNA in an expression vector is an alternative strategy to stably
suppress gene expression, and such constructs with well defined initiation and
termination sites have been used to produce various small RNA species that
inhibit the expression of genes with diverse functions in mammalian cell lines
(39). The conditional
alteration of gene expression by the use of an shRNA expression vector holds
potential promise for therapeutic approaches for silencing disease-causing
genes provided that appropriate extracellular and intracellular nucleic acid
delivery systems (vector systems) are available that offer efficient vehicles
for stable complexation and protection of the nucleic acid. To reach the
targeted tissue, vectors need to overcome a number of extracellular and
intracellular barriers. Systemic targeting by viral vectors toward the desired
tissue is difficult because the host immune responses activate viral
clearance. Systemic administration of a large amount of adenovirus
(e.g. into the liver) can be a serious health hazard that even caused
the death of one patient
(40).Nonviral vectors, such as positively charged PEI complexes, mediate
unspecific interactions with non-target cells and blood components, which
results in the rapid clearance from the circulation. These unfavorable effects
can be minimized by “shielding” of the positive surface charge of
the vectors with polyethylene glycol (PEG). PEGylation of PEI polyplexes can
prevent the systemic degradation of the plasmid DNA and reduce the toxicity of
polyplexes (41). To increase
the transfection efficiency of the shielded particles (plasmid DNA/PEG-PEI),
different targeting ligands, such as peptide, growth factors and proteins, or
antibodies, have been incorporated into the vectors
(42). One such targeting
ligands is transferrin (Tf), an iron-transporting protein that is recognized
by Tf receptors (Tf-R) present at high levels in the tumor cells
(42,
43). In contrast, in
nonproliferating cells, expression of Tf-R is low or undetectable. Association
of Tf to polyplexes significantly enhances transfection efficiency by
promoting the internalization of polyplexes (plasmid DNA/Tf-PEG-PEI
(designated as nanoparticles throughout this study)) in dividing and
nondividing cells (42). After
cellular association of nanoparticles to the target cells, particles are
internalized by receptor-mediated endocytosis
(42).
Fig. 1 illustrates that the
uptake of nanoparticles carrying multiple functional domains (surface
shielding particles Tf-PEG-PEI, tissue-specific promoter-driven Cre
recombinase, and conditionally silenced plasmid) can overcome the
intracellular barriers for successful delivery of the shRNA gene.Open in a separate windowFIGURE 1.Schematic illustration of cellular uptake of plasmid DNA/Tf-PEG-PEI
(nanoparticles) polyplexes, their shielding from nonspecific interaction, and
mechanism of action of shRNA. Internalization of PEG-shielded and
Tf-R-targeted polyplexes into target cells occurs by receptor-mediated
endocytosis after association of polyplex ligand Tf to Tf-R present on the
target cell plasma membrane. Internalized particles are trafficked to
endosomes followed by endosomal release of the particles and/or the nucleic
acid into cytoplasm. Released siRNA will be induced to RNA-induced silencing
complex and will be guided for cleavage of complementary target mRNA in the
cytoplasm. siRNA (antisense) guide strand will direct the targeted RNAs to be
cleaved by RNA endonuclease. Finally plasmid/Tf-PEG-PEI-nanoparticles delivery
in the target cell shows reporter gene expression and activity.We tested the effects of CD44v6 shRNA in vivo in a mouse model of
human familial adenomatous polyposis (FAP) where adenomatous polyposis coli
gene (Apc) is mutated. Most FAP patients carry truncation mutations
in the N-terminal half (44).
Several mouse models of FAP were constructed either by chemical mutagenesis of
the Apc gene at codon 850 (Apc Min/+ mouse) or by homologous
recombination in embryonic stem cells (knock-out strains) such as Apc
1638N, Apc 1638T, Apc 1309, Apc Δ716, and
Apc Δ474 (45).
Germ line mutagenesis of C57BL/6J (B6) males was done by N-ethyl
N-nitrosourea, and the offspring of mutated B6 × AKR/J (AKR)
females yielded mice that carry 100% of an autosomal dominant mutation at
codon 850 in the Apc gene. These five strains have mutated
Apc alleles that encode truncated Apc proteins of 850, 1638, 1309,
716, and 474 amino acids, respectively. These variable truncated products form
dimers with wild-type Apc protein and show variable dominant negative
activities. As a result, the number of polyps varies in different strains. For
example, the number of polyps at 16 weeks of age are ∼100 in Apc
Min/+, Apc Δ474, and Apc 1309 mice. A much greater
number of polyps (300-400) are found in Apc Δ716 mice, whereas
much fewer polyps (∼10) are found in Apc 638N mice
(46). These mutations in the
Apc allele and the phenotype variability observed in FAP patients
allowed the establishment of genotype-phenotype correlations at the
Apc locus resulting in multiple intestinal adenomas throughout the
length of the small and large intestine
(47). Interestingly, most
polyps are found in the small intestine, although a small but significant
number of polyps develop in the colon, a phenotype different from human FAP.
Despite this caveat, the Apc Min/+ mouse offers the prospect to study
intestinal tumors that are of the same genetic background as the host. Strong
up-regulations of CD44, including both CD44s and CD44v6 encoded epitopes, and
of Cox-2 were observed in aberrant crypt foci in Apc Min/+
mice (48,
49). Furthermore, CD44v
proteins can form multimeric complexes in the plasma membrane, which
dramatically enhances their HA binding capacity
(11). Moreover, our results
with Apc Min/+ mice confirm earlier findings that Tf-R is present at
high levels in the tumor cells
(42), which is crucial for
active targeting of Tf-R by Tf-mediated CD44v6 shRNA delivery in tumor cells.
For these reasons, the Apc Min/+ mouse model can be used for
modulating adenoma growth by using HA-CD44v6 interaction antagonists as
therapeutic agents in vivo.In this study we tested whether HA regulates Cox-2 via its effects
on a CD44v6 → ErbB2 → Cox-2 axis in colon cancer cells.
More importantly we provide evidence that systemic application of
(pSico-CD44v6 shRNA plus pFabpl-Cre)/nanoparticles in the
Apc Min/+ mouse model reduces intestinal tumor growth by perturbing
CD44v6 containing isoform expression. 相似文献
3.
M. BODO P. CARINCI T. BARONI E. BECCHETTI C. BELLUCCI F. PEZZETTI M. GIAMMARIOLI G. STABELLINI N. ARENA 《Cell biology international》1996,20(3):177-185
Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α2more than a1 chain production.3H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton. 相似文献
4.
5.
Stephen H. Prior Todd S. Byrne Dorota Tokmina-Roszyk Gregg B. Fields Steven R. Van Doren 《The Journal of biological chemistry》2016,291(15):7888-7901
Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å2 of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling. 相似文献
6.
THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS
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Arthur Karlin 《The Journal of cell biology》1965,25(2):159-169
Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed. 相似文献
7.
8.
Roger D. Meicenheimer 《American journal of botany》1992,79(8):914-920
The stem unit is defined as the smallest portion of a stem that can duplicate the stem in toto through regular rotations and dilations over successive plastochrons. In stems exhibiting k(m, n) contact parastichy phyllotaxis, the stem unit is delimited vertically and tangentially by the boundaries of four successive leaf primordia along the m-, n-, and (m + n)- parastichies and radially by these boundaries extended to the centroid of the stem. With the stem unit concept, node refers only to the region of actual leaf insertion, rather than the entire transverse level of insertion. Many of the conflicting and complicating aspects of the traditional node-intemode subdivisions of stems are demonstrated, and the utility of the stem unit in circumventing these is illustrated. The stem unit is proposed as a more useful analytic subunit of the stem with which to examine stem growth and tissue differentiation processes than the more traditional node-intemode subdivisions of stems. 相似文献
9.
10.
Irreversible proliferation and cornification of the mouse vaginal epithelium were induced by 10 daily injections of 20μg estradiol-17β starting on the day of birth. Development of the irreversible vaginal epithelium during the period of estrogen injections in early postnatal life was observed under light and electron microscopes. Small electron-dense cells (A-cells) in clusters were present in the columnar vaginal epithelium of newborn mice. A-cells were proliferated by 2 daily estrogen injections. At the sites of A-cell clumps, large electron-dense cells (B-cells) characterized by long winding cytoplasmic processes appeared in mice given 3 daily injections, forming nodules which then fused together to form a layer under the columnar epithelium after 4 daily injections. In mice given 7 daily injections, the primary epithelium was shed by the superficial cornification of the newly formed layer. The B-cell membrane bore fewer desmosomes than in the basal and intermediary cells of the vaginal epithelium of ovariectomized ‘normal’ adult mice after 5 daily injections of 100μg estradiol-17β. Hyperplastic epithelial downgrowths in old ovariectomized mice given neonatal estrogen injections contained another type of cells with reduced density which formed much fewer processes and only a few desmosomes (C-cells). 相似文献
11.
12.
13.
14.
The ubiquitously expressed reduced folate carrier (RFC) is the major
transport system for folate cofactors in mammalian cells and tissues. Previous
considerations of RFC structure and mechanism were based on the notion that
RFC monomers were sufficient to mediate transport of folate and antifolate
substrates. The present study examines the possibility that human RFC (hRFC)
exists as higher order homo-oligomers. By chemical cross-linking, transiently
expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional
cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting,
hRFC species with molecular masses of hRFC homo-oligomers were identified.
Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were
co-immunoprecipitated from both membrane particulate and surface-enriched
membrane fractions, indicating that oligomeric hRFC is expressed at the cell
surface. By co-expression of wild type and inactive mutant S138C hRFCs,
combined with surface biotinylation and confocal microscopy, a
dominant-negative phenotype was demonstrated involving greatly decreased cell
surface expression of both mutant and wild type carrier caused by impaired
intracellular trafficking. For another hRFC mutant (R373A), expression of
oligomeric wild type-mutant hRFC was accompanied by a significant and
disproportionate loss of wild type activity unrelated to the level of surface
carrier. Collectively, our results demonstrate the existence of hRFC
homo-oligomers. They also establish the likely importance of these higher
order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the
synthesis of nucleotide precursors, serine, and methionine in one-carbon
transfer reactions (1). Because
mammals cannot synthesize folates de novo, cellular uptake of these
derivatives is essential for cell growth and tissue regeneration
(2,
3). Folates are hydrophilic
anionic molecules that do not cross biological membranes by diffusion alone,
so it is not surprising that sophisticated membrane transport systems have
evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier
(RFC)2 is widely
considered to be the major transport system for folate co-factors in mammalian
cells and tissues (3,
4). RFC plays a generalized
role in folate transport and provides specialized tissue functions such as
transport across the basolateral membrane of renal proximal tubules
(5), transplacental transport
of folates (6), and folate
transport across the blood-brain barrier
(7), although the contribution
of RFC to intestinal absorption of folates remains controversial
(8,
9). Loss of RFC expression or
function portends potentially profound physiologic and developmental
consequences associated with folate deficiency
(10). RFC is also a major
transporter of antifolate drugs used for cancer chemotherapy such as
methotrexate (Mtx), pemetrexed, and raltitrexed
(4). Loss of RFC expression or
synthesis of mutant RFC protein in tumor cells results in antifolate
resistance caused by incomplete inhibition of cellular enzyme targets and low
levels of antifolate substrate for polyglutamate synthesis
(4,
11).Reflecting its particular physiologic and pharmacologic importance,
interest in RFC structure and function has been high. Since 1994, when murine
RFC was first cloned (12),
application of state-of-the-art molecular biology and biochemistry methods for
characterizing polytopic membrane proteins has led to a progressively detailed
picture of the molecular structure of the carrier, including its membrane
topology, N-glycosylation, functionally or structurally important
domains and amino acids, and packing of α-helix transmembrane domains
(TMDs) (4,
13). Although no crystal
structure for RFC has yet been reported, a detailed homology model for human
RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol
3-phosphate/inorganic phosphate antiporter GlpT was generated
(13,
14) that permits testing of
hypotheses related to hRFC structure and mechanism in a manner not previously
possible.Considerations of hRFC structure and mechanism to date have all been based
on the notion that a single 591-amino acid hRFC molecule is sufficient to
mediate concentrative uptake of folate and antifolate substrates. However, a
growing literature suggests that quaternary structure involving the formation
of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly
an important feature of the structure and function of many membrane
transporters
(15-18).
For major facilitator superfamily proteins, both monomeric (e.g.
LacY, GlpT, UhpT, and GLUT3)
(19-22)
and oligomeric (e.g. LacS, AE1, GLUT1, and TetA)
(23-28)
structures have been reported, establishing the lack of a clear structural
consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a
homo-oligomeric species composed of multiple hRFC monomers. Based on results
with an assortment of biochemical methods with wt and a collection of mutant
hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but
also establish the probable importance of these higher order structures to
intracellular trafficking and carrier function. 相似文献
15.
16.
17.
Atya Aziz John F. Hess Madhu S. Budamagunta Paul G. FitzGerald John C. Voss 《The Journal of biological chemistry》2009,284(11):7330-7338
18.
Irina A. Rodionova Harmon J. Zuccola Leonardo Sorci Alexander E. Aleshin Marat D. Kazanov Chen-Ting Ma Eduard Sergienko Eric J. Rubin Christopher P. Locher Andrei L. Osterman 《The Journal of biological chemistry》2015,290(12):7693-7706
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the
biosynthesis of the NAD cofactor, which has been implicated as a target for
developing new antimycobacterial therapies. Here we report the crystal structure
of Mycobacterium tuberculosis NadD (MtNadD) at
a resolution of 2.4 Å. A remarkable new feature of the
MtNadD structure, compared with other members of this
enzyme family, is a 310 helix that locks the active site in an
over-closed conformation. As a result, MtNadD is rendered
inactive as it is topologically incompatible with substrate binding and
catalysis. Directed mutagenesis was also used to further dissect the structural
elements that contribute to the interactions of the two MtNadD
substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN).
For inhibitory profiling of partially active mutants and wild type
MtNadD, we used a small molecule inhibitor of
MtNadD with moderate affinity
(Ki ∼ 25 μm) and
antimycobacterial activity (MIC80) ∼ 40–80
μm). This analysis revealed interferences with some of the
residues in the NaMN binding subsite consistent with the competitive inhibition
observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic
analysis of MtNadD suggests that ATP must first bind to allow
efficient NaMN binding and catalysis. This sequential mechanism is consistent
with the requirement of transition to catalytically competent (open)
conformation hypothesized from structural modeling. A possible physiological
significance of this mechanism is to enable the down-regulation of NAD synthesis
under ATP-limiting dormancy conditions. These findings point to a possible new
strategy for designing inhibitors that lock the enzyme in the inactive
over-closed conformation. 相似文献
19.