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91.
Zilberberg L Todorovic V Dabovic B Horiguchi M Couroussé T Sakai LY Rifkin DB 《Journal of cellular physiology》2012,227(12):3828-3836
Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin‐1 and fibrillin‐2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril‐associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF‐β binding protein 1 (LTBP‐1), ‐3 and ‐4; the three LTBPs that form a complex with TGF‐β. In Fbn1?/? ascending aortas and lungs, LTBP‐3 and LTBP‐4 are not incorporated into a matrix lacking fibrillin‐1 microfibrils, whereas LTBP‐1 is still deposited. In addition, in cultures of Fbn1?/? smooth muscle cells or lung fibroblasts, LTBP‐3 and LTBP‐4 are not incorporated into a matrix lacking fibrillin‐1 microfibrils, whereas LTBP‐1 is still deposited. Fibrillin‐2 is not involved in the deposition of LTBP‐1 in Fbn1?/? extracellular matrix as cells deficient for both fibrillin‐1 and fibrillin‐2 still incorporate LTBP‐1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1?/? cells abrogates the deposition of LTBP‐1. Together, these data indicate that LTBP‐3 and LTBP‐4 association with the matrix depends on fibrillin‐1 microfibrils, whereas LTBP‐1 association depends on a fibronectin network. J. Cell. Physiol. 227: 3828–3836, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
92.
Ezernitchi AV Vaknin I Cohen-Daniel L Levy O Manaster E Halabi A Pikarsky E Shapira L Baniyash M 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(7):4763-4772
T cell AgR zeta chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; zeta chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1(+)Mac-1(+) myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their zeta expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer. 相似文献
93.
94.
Abu-Abied M Golomb L Belausov E Huang S Geiger B Kam Z Staiger CJ Sadot E 《The Plant journal : for cell and molecular biology》2006,48(3):367-379
Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves. 相似文献
95.
Two recent studies have provided the most complete screen for protein complexes in yeast to date, in which partners were identified for approximately half of the proteome. A comparison shows that these two datasets are complementary. In addition, one of the analyses points to a modular organization of the cellular protein network. These data will prove useful in defining principles and trends that arise when combining large-scale datasets of different natures, and in deriving properties of protein machines in cellular systems. 相似文献
96.
Lior Ungar Nir Yosef Yael Sela Roded Sharan Eytan Ruppin Martin Kupiec 《Nucleic acids research》2009,37(12):3840-3849
Telomeres are structures composed of repetitive DNA and proteins that protect the chromosomal ends in eukaryotic cells from fusion or degradation, thus contributing to genomic stability. Although telomere length varies between species, in all organisms studied telomere length appears to be controlled by a dynamic equilibrium between elongating mechanisms (mainly addition of repeats by the enzyme telomerase) and nucleases that shorten the telomeric sequences. Two previous studies have analyzed a collection of yeast deletion strains (deleted for nonessential genes) and found over 270 genes that affect telomere length (Telomere Length Maintenance or TLM genes). Here we complete the list of TLM by analyzing a collection of strains carrying hypomorphic alleles of most essential genes (DAmP collection). We identify 87 essential genes that affect telomere length in yeast. These genes interact with the nonessential TLM genes in a significant manner, and provide new insights on the mechanisms involved in telomere length maintenance. The newly identified genes span a variety of cellular processes, including protein degradation, pre-mRNA splicing and DNA replication. 相似文献
97.
Jayita Guhaniyogi Istvan Sohar Kalyan Das Ann M. Stock Peter Lobel 《The Journal of biological chemistry》2009,284(6):3985-3997
Late infantile neuronal ceroid lipofuscinosis is a fatal childhood
neurological disorder caused by a deficiency in the lysosomal protease
tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian
member of the S53 family of serine proteases, a group characterized by a
subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum.
TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is
proteolytically processed into the active enzyme after exposure to low pH
in vitro or targeting to the lysosome in vivo. In this
study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing
four Asn-linked N-acetylglucosamines that is indistinguishable from
fully glycosylated TPP1 in terms of autocatalytic processing of the proform
and enzymatic properties of the mature protease. The crystal structure of
deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large
151-residue C-shaped prodomain makes extensive contacts as it wraps around the
surface of the catalytic domain with the two domains connected by a 24-residue
flexible linker that passes through the substrate-binding groove. The
proenzyme structure reveals suboptimal catalytic triad geometry with its
propiece linker partially blocking the substrate-binding site, which together
serve to prevent premature activation of the protease. Finally, we have
identified numerous processing intermediates and propose a structural model
that explains the pathway for TPP1 activation in vitro. These data
provide new insights into TPP1 function and represent a valuable resource for
constructing improved TPP1 variants for treatment of late infantile neuronal
ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis
(LINCL)3 (OMIM number
204500) is a neurodegenerative lysosomal storage disease of childhood that
presents typically between the ages of 2 and 4 years with the onset of
seizures. Disease progression is reflected by blindness, dementia, mental
retardation, and an increase in the severity of seizures. LINCL is always
fatal, and the life span of patients is typically 6-15 years. LINCL is caused
by mutations in TPP1 (previously named CLN2, for ceroid
lipofuscinosis neuronal type 2 gene)
(1), which normally encodes a
lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9)
(2,
3).There is currently no treatment of demonstrated efficacy for LINCL, but
promising progress is being made in some directions. Proof-of-principle for
virus-mediated gene therapy has been established in a mouse model of LINCL,
with a significant improvement in disease phenotype achieved with the use of
adeno-associated virus vectors expressing TPP1
(4-7).
Affected children have also been treated with adeno-associated virus vectors,
although it is too soon to determine whether significant clinical benefits
have been achieved in these early trials
(8). Enzyme replacement
therapy, an approach that has proven successful in a number of other lysosomal
storage diseases, has also been investigated in LINCL. Purified recombinant
human TPP1 that contains the mannose 6-phosphate lysosomal targeting
modification can be taken up by LINCL fibroblasts where it degrades storage
material (9), and the protein
has been introduced into the cerebrospinal fluid of the LINCL mouse model via
intraventricular injection, resulting in significant uptake into the brain and
some correction of neuropathology
(10).For therapeutic approaches that rely upon replacing a mutant gene product
with a functional protein via recombinant methods, e.g. gene and
enzyme replacement therapy, a thorough understanding of the biological and
biophysical properties of the protein in question are essential for success.
Thus, for LINCL, considerable effort has been directed toward the
investigation of TPP1, and as a result, this is a well characterized enzyme at
the functional and molecular levels (reviewed in Refs.
11,
12). TPP1 encodes a
563-residue preproprotein with a cleavable N-terminal 19-residue signal
sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes
proteolytic cleavage in the lysosome, converting the zymogen to an active,
mature protease (residues 196-563)
(1). Studies on purified
pro-TPP1 demonstrate that maturation is autocatalytic in vitro
(13,
14) but may involve other
proteases in vivo
(15). TPP1 is glycosylated,
and its N-linked oligosaccharides have been implicated in maturation,
activity, targeting, and stability of the processed enzyme
(16,
17).TPP1 is a serine protease
(14) that possesses two
catalytic functions as follows: a primary tripeptidyl exopeptidase activity
with a pH optimum of ∼5.0 that catalyzes the sequential release of
tripeptides from the unsubstituted N termini of substrates
(18), and a much weaker
endoproteolytic activity with a pH optimum of ∼3.0
(19). TPP1 exhibits broad
substrate specificity (20) and
is the only mammalian member of the S53 sedolisin family (reviewed in Ref.
21), which includes a number
of unusual bacterial serine peptidases
(22). High resolution crystal
structures of both free and inhibitor-bound complexes have been determined for
three bacterial members of this family (sedolisin
(23-26),
kumamolisin (27,
28), and kumamolisin-As
(29,
30)), and for one
(kumamolisin), the structure of a mutant, inactive precursor form has also
been obtained (28). These
proteins share a common subtilisin-like fold, an octahedrally coordinated
calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp
(SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin
(31,
32). Chemical modification
studies of TPP1 have revealed that Ser475 is the active site
nucleophile (14). Modeling
studies suggest that Glu272 and Asp276 complete the
catalytic triad and that Asp360 is homologous to the conserved Asn
in the subtilisin family in its role in stabilization of the oxyanion of the
tetrahedral intermediate during catalysis
(33). Site-directed
mutagenesis studies are consistent with these conclusions
(14,
34).A detailed understanding of the tertiary structure of TPP1 may have
implications for developing or improving therapeutic strategies. First, a high
resolution model would provide the basis for targeted protein engineering
efforts to design TPP1 derivatives with increased half-life prior to and/or
upon delivery to the lysosome. Successful creation of a longer lived TPP1
molecule could significantly enhance gene or enzyme replacement approaches to
LINCL. Second, a structural model for TPP1 could be valuable in designing
derivatives tagged with protein transduction domains to facilitate crossing of
the blood-brain barrier for delivery to the central nervous system from the
bloodstream. In this study, we present the crystal structure of the proform of
human TPP1 at 1.85 Å resolution. This model provides novel insights into
the structural basis for the pH-induced auto-activation of the proform of
TPP1. A structure of glycosylated pro-TPP1 has been independently determined,
displaying features similar to those of deglycosylated
TPP1.4 相似文献
98.
Robert N. Ono Gerhard Sengle Noe L. Charbonneau Valerie Carlberg Hans Peter B?chinger Takako Sasaki Sui Lee-Arteaga Lior Zilberberg Daniel B. Rifkin Francesco Ramirez Mon-Li Chu Lynn Y. Sakai 《The Journal of biological chemistry》2009,284(25):16872-16881
Latent transforming growth factor (TGF) β-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFβ. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit “exquisite specificities,” a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.Fibrillin microfibrils are ubiquitous structural elements in the connective tissue. Fibrillin microfibrils provide organs with tissue-specific architectural frameworks designed to support the mature functional integrity of the particular organ. In addition, fibrillin microfibrils contribute to proper developmental patterning of organs by targeting growth factors to the right location in the extracellular matrix (1, 2).Molecules of fibrillin-1 (3), fibrillin-2 (4, 5), and fibrillin-3 (6) polymerize to form the backbone structure of microfibrils. Latent TGFβ-binding protein (LTBP)3-1 associates with fibrillin microfibrils in the perichondrium and in osteoblast cultures (7, 8), and LTBP-1 and LTBP-4 interact with fibrillin (9). Other proteins associated with fibrillin microfibrils include the fibulins (10, 11), microfibril-associated glycoprotein-1 and -2 (12, 13), decorin (14), biglycan (15), versican (16), and perlecan (17). It is likely that one function of these associated extracellular matrix molecules is to connect the fibrillin microfibril scaffold to other architectural elements in tissue- and organ-specific patterns.In addition to performing architectural functions, fibrillins bind directly to prodomains of bone morphogenetic proteins and growth and differentiation factors (18, 19) and LTBPs bring with them the small latent TGFβ complex (20), suggesting that the microfibril scaffold may position, concentrate, and control growth factor signaling. Studies of fibrillin-1 (Fbn1) and fibrillin-2 (Fbn2) mutant mice demonstrate that loss of fibrillins results in phenotypes associated with dysregulated TGFβ (21–23) or bone morphogenetic protein (24) signaling. Microfibril-associated glycoprotein-1 (Magp-1) null mice reveal phenotypes that may also be related to abnormal TGFβ signaling (25).In a previous study (9), we determined that the binding site for LTBP-1 and -4 is contained within a specific four-domain region of fibrillin-1. In this study, we performed additional experiments to more precisely define the LTBP binding site. At the same time, we compared binding of fibulins to fibrillin, because the region in fibrillin-1 that was suggested to contain the fibulin binding site (11) was very close to our region of interest for LTBP binding. Our results demonstrate that LTBPs and fibulins compete for binding to fibrillin-1. However, the proteins tested (LTBP-1, LTBP-4, fibulin-2, fibulin-4, and fibulin-5) displayed “exquisite specificities” in their interactions with fibrillin-1.To test the potential significance of these interactions with fibrillin-1, we investigated matrix incorporation of LTBPs in cell cultures obtained from wild type, Fbn1 null, Fbn2 null, fibulin-2 (Fbln-2) null, and fibulin-4 (Fbln-4) null mice. In addition, we examined the distribution of LTBPs in Fbn1 null and Fbn2 null mice. 相似文献
99.
Robert K. Bradley Adam Roberts Michael Smoot Sudeep Juvekar Jaeyoung Do Colin Dewey Ian Holmes Lior Pachter 《PLoS computational biology》2009,5(5)
We describe a new program for the alignment of multiple biological sequences that is both statistically motivated and fast enough for problem sizes that arise in practice. Our Fast Statistical Alignment program is based on pair hidden Markov models which approximate an insertion/deletion process on a tree and uses a sequence annealing algorithm to combine the posterior probabilities estimated from these models into a multiple alignment. FSA uses its explicit statistical model to produce multiple alignments which are accompanied by estimates of the alignment accuracy and uncertainty for every column and character of the alignment—previously available only with alignment programs which use computationally-expensive Markov Chain Monte Carlo approaches—yet can align thousands of long sequences. Moreover, FSA utilizes an unsupervised query-specific learning procedure for parameter estimation which leads to improved accuracy on benchmark reference alignments in comparison to existing programs. The centroid alignment approach taken by FSA, in combination with its learning procedure, drastically reduces the amount of false-positive alignment on biological data in comparison to that given by other methods. The FSA program and a companion visualization tool for exploring uncertainty in alignments can be used via a web interface at http://orangutan.math.berkeley.edu/fsa/, and the source code is available at http://fsa.sourceforge.net/. 相似文献
100.
The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity.We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation.