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Biomedical subjects

A Godzik

Publications and source records attributed to A Godzik.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin.

Identification of expanding roles for matrix metalloproteinases (MMPs) in complex regulatory processes of tissue remodelling has stimulated the search for genes encoding proteinases with unique functions, regulation and expression patterns. By using a novel cloning strategy, we identified three previously unknown human MMPs, i.e. MMP-21, MMP-26 and MMP-28, in comprehensive gene libraries. The present study is focused on the gene and the protein of a novel MMP, MMP-26. Our findings show that MMP-26 is specifically expressed in cancer cells of epithelial origin, including carcinomas of lung, prostate and breast. Several unique structural and regulatory features, including an unusual 'cysteine-switch' motif, discriminate broad-spectrum MMP-26 from most other MMPs. MMP-26 efficiently cleaves fibrinogen and extracellular matrix proteins, including fibronectin, vitronectin and denatured collagen. Protein sequence, minimal modular domain structure, exon-intron mapping and computer modelling demonstrate similarity between MMP-26 and MMP-7 (matrilysin). However, substrate specificity and transcriptional regulation, as well as the functional role of MMP-26 and MMP-7 in cancer, are likely to be distinct. Despite these differences, matrilysin-2 may be a suitable trivial name for MMP-26. Our observations suggest an important specific function for MMP-26 in tumour progression and angiogenesis, and confirm and extend the recent findings of other authors [Park, Ni, Gerkema, Liu, Belozerov and Sang (2000) J. Biol. Chem. 275, 20540--20544; Uría and López-Otín (2000) Cancer Res. 60, 4745--4751; de Coignac, Elson, Delneste, Magistrelli, Jeannin, Aubry, Berthier, Schmitt, Bonnefoy and Gauchat (2000) Eur. J. Biochem. 267, 3323--3329].

Amino Acid Sequence↗
PubMed2001

TUCAN, an antiapoptotic caspase-associated recruitment domain family protein overexpressed in cancer.

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulated CARD-containing antagonist of caspase nine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Surface map comparison: studying function diversity of homologous proteins.

A simplified protein surface cartography approach has been developed to assist in the analysis of surface features in homologous families, and thus to predict conservation or divergence of protein functions and protein-protein interaction patterns. A spherical approximation of protein surface was used, with a focus on charged and hydrophobic residues. The resulting surface map allows for qualitative analysis and comparison of surfaces of proteins, but can also be used to define a simple numerical measure of map similarity between two or more proteins. The latter was shown to be useful for function based classifications within large protein families. Surface map analysis was tested on several test cases: haemoglobins, death domains and TRAF domains. It was shown that surface map comparison allows a better function prediction than general sequence analysis methods and can reproduce known examples of functional variation within a divergent group of proteins. In another example, we predict novel, unexpected sets of common functional properties for seemingly distant members of a large group of divergent proteins. The method was also shown to be robust enough to allow using protein models from comparative modelling instead of experimental structures.

Animals↗
PubMed2001

Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak.

A novel human member of the Bcl-2 family was identified, Bcl-B, which is closest in amino acid sequence homology to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2 homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted carboxyl-terminal transmembrane (TM) domain. The BCL-B mRNA is widely expressed in adult human tissues. The Bcl-B protein binds Bcl-2, Bcl-X(L), and Bax but not Bak. In transient transfection assays, Bcl-B suppresses apoptosis induced by Bax but not Bak. Deletion of the TM domain of Bcl-B impairs its association with intracellular organelles and diminishes its anti-apoptotic function. Bcl-B thus displays a unique pattern of selectivity for binding and regulating the function of other members of the Bcl-2 family.

Amino Acid Sequence↗
PubMed2001

A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains.

We have identified three new tumor necrosis factor-receptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.

Amino Acid Sequence↗
PubMed2001

CLAN, a novel human CED-4-like gene.

Proteins governing cell death form the basis of many normal processes and contribute to the pathogenesis of many diseases when dysregulated. Here we report the cloning of a novel human CED-4-like gene, CLAN, and several of its alternatively spliced isoforms. These caspase-associated recruitment domain (CARD)-containing proteins are expressed at varying degrees in normal human tissues and may contribute to a number of intracellular processes including apoptosis, cytokine processing, and NF-kappa B activation. The CARD of the CLAN proteins binds a number of other CARD-containing proteins including caspase-1, BCL10, NOD2, and NAC. Once their physiologic functions are uncovered, CLAN proteins may prove to be valuable therapeutic targets.

Amino Acid Sequence↗
PubMed2001

Clustering of highly homologous sequences to reduce the size of large protein databases.

We present a fast and flexible program for clustering large protein databases at different sequence identity levels. It takes less than 2 h for the all-against-all sequence comparison and clustering of the non-redundant protein database of over 560,000 sequences on a high-end PC. The output database, including only the representative sequences, can be used for more efficient and sensitive database searches.

Algorithms↗
PubMed2000

A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis.

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.

Amino Acid Sequence↗
PubMed2000

Bcl-G, a novel pro-apoptotic member of the Bcl-2 family.

A new member of the Bcl-2 family was identified, Bcl-G. The human BCL-G gene consists of 6 exons, resides on chromosome 12p12, and encodes two proteins through alternative mRNA splicing, Bcl-G(L) (long) and Bcl-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. Bcl-G(L) and Bcl-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. Among the Bcl-2 homology (BH) domains previously recognized in Bcl-2 family proteins, the BH3 domain is found in both Bcl-G(L) and Bcl-G(S), but only the longer Bcl-G(L) protein possesses a BH2 domain. Bcl-G(L) mRNA is expressed widely in adult human tissues, whereas Bcl-G(S) mRNA was found only in testis. Overexpression of Bcl-G(L) or Bcl-G(S) in cells induced apoptosis although Bcl-G(S) was far more potent than Bcl-G(L). Apoptosis induction by Bcl-G(S) depended on the BH3 domain and was suppressed by coexpression of anti-apoptotic Bcl-X(L) protein. Bcl-X(L) also coimmunoprecipitated with Bcl-G(S) but not with mutants of Bcl-G(S) in which the BH3 domain was deleted or mutated or with Bcl-G(L). Bcl-G(S) was predominantly localized to cytosolic organelles, whereas Bcl-G(L) was diffusely distributed throughout the cytosol. A mutant of Bcl-G(L) in which the BH2 domain was deleted displayed increased apoptotic activity and coimmunoprecipitated with Bcl-X(L), suggesting that the BH2 domain autorepresses Bcl-G(L).

Alternative Splicing↗
PubMed2000

Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms.

Genetic analysis of programmed cell death in Drosophila reveals many similarities with mammals. Heretofore, a missing link in the fly has been the absence of any Bcl-2/Bax family members, proteins that function in mammals as regulators of mitochondrial cytochrome c release. A Drosophila homologue of the human killer protein Bok (DBok) was identified. The predicted structure of DBok is similar to pore-forming Bcl-2/Bax family members. DBok induces apoptosis in insect and human cells, which is suppressible by anti-apoptotic human Bcl-2 family proteins. A caspase inhibitor suppressed DBok-induced apoptosis but did not prevent DBok-induced cell death. Moreover, DBok targets mitochondria and triggers cytochrome c release through a caspase-independent mechanism. These characteristics of DBok reveal evolutionary conservation of cell death mechanisms in flies and humans.

Amino Acid Sequence↗
PubMed2000

AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC.

AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli. AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction. Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center. Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F[208-521]) and the 2Tr-like N-terminal domain (F[1-202]) have been addressed. Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties. Electron transfer between F[208-521] and F[1-202] is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F[1-202]. TrR is nearly as efficient at F[1-202] reduction as is F[208-521], although neither the latter fragment, nor intact AhpF, can reduce Tr. An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F[1-202], and not F[208-521], is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC.

Amino Acid Sequence↗
PubMed2000

ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4.

Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N-ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964-10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis.

Adenosine Triphosphate↗
PubMed2000

The Drosophila tumor necrosis factor receptor-associated factor-1 (DTRAF1) interacts with Pelle and regulates NFkappaB activity.

A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family was identified in Drosophila. DTRAF1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of DTRAF1 binding to different members of the human TNF receptor family showed that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-beta receptor. DTRAF1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, DTRAF1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, DTRAF1 did not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increased NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction was partially inhibited by DTRAF1. Mutants of DTRAF1 lacking the N-terminal region inhibited NFkappaB induction by either TRAF2 or TRAF6. DTRAF1 specifically associated with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and DTRAF1 individually were unable to induce NFkappaB in a human cell line, co-expression of Pelle and DTRAF1 resulted in significant NFkappaB activity. Interactions of DTRAF1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution.

Amino Acid Sequence↗
PubMed2000

BAR: An apoptosis regulator at the intersection of caspases and Bcl-2 family proteins.

Two major pathways for induction of apoptosis have been identified-intrinsic and extrinsic. The extrinsic pathway is represented by tumor necrosis factor family receptors, which utilize protein interaction modules known as death domains and death effector domains (DEDs) to assemble receptor signaling complexes that recruit and activate certain caspase-family cell death proteases, namely procaspases-8 and -10. The intrinsic pathway for apoptosis involves the participation of mitochondria, which release caspase-activating proteins. Bcl-2 family proteins govern this mitochondria-dependent apoptosis pathway, with proteins such as Bax functioning as inducers and proteins such as Bcl-2 and Bcl-X(L) serving as suppressors of cell death. An apoptosis regulator, BAR, was identified by using a yeast-based screen for inhibitors of Bax-induced cell death. The BAR protein contains a SAM domain, which is required for its interactions with Bcl-2 and Bcl-X(L) and for suppression of Bax-induced cell death in both mammalian cells and yeast. In addition, BAR contains a DED-like domain responsible for its interaction with DED-containing procaspases and suppression of Fas-induced apoptosis. Furthermore, BAR can bridge procaspase-8 and Bcl-2 into a protein complex. The BAR protein is anchored in intracellular membranes where Bcl-2 resides. BAR therefore may represent a scaffold protein capable of bridging two major apoptosis pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2000

From fold to function predictions: an apoptosis regulator protein BID.

With the rapidly increasing pace of genome sequencing projects and the resulting flood of predicted amino acid sequences of uncharacterized proteins, protein sequence analysis, and in particular, protein structure prediction is quickly gaining in importance. Prediction algorithms can be used for preliminary annotation of newly sequenced proteins and, at least in some cases, provide insights into their function and specific mode of action. Such annotations for several microbial genomes were performed by several groups and placed in public domain for evaluation. An example presented in this work comes from a related project of structural and functional predictions for proteins involved in the process of controlled cell death (apoptosis). The BID protein belongs to an important class of regulators of apoptosis identified by short sequence motifs. Here, several fold prediction methods are used to build a series of three-dimensional models. Structure analysis of the models with reference to the biological data available allows selection of the most appropriate model. It is found that the most likely structural model of BID is built on the structure of Bcl-X(L). The model is discussed in terms of experimental data on specific proteolytic cleavage of BID and its effect on BID interactions with other proteins and membranes.

Algorithms↗
PubMed2000

Saturated BLAST: an automated multiple intermediate sequence search used to detect distant homology.

MOTIVATION: Two proteins can have a similar 3-dimensional structure and biological function, but have sequences sufficiently different that traditional protein sequence comparison algorithms do not identify their relationship. The desire to identify such relations has led to the development of more sensitive sequence alignment strategies. One such strategy is the Intermediate Sequence Search (ISS), which connects two proteins through one or more intermediate sequences. In its brute-force implementation, ISS is a strategy that repetitively uses the results of the previous query as new search seeds, making it time-consuming and difficult to analyze. RESULTS: Saturated BLAST is a package that performs ISS in an efficient and automated manner. It was developed using Perl and Perl/Tk and implemented on the LINUX operating system. Starting with a protein sequence, Saturated BLAST runs a BLAST search and identifies representative sequences for the next generation of searches. The procedure is run until convergence or until some predefined criteria are met. Saturated BLAST has a friendly graphic user interface, a built-in BLAST result parser, several multiple alignment tools, clustering algorithms and various filters for the elimination of false positives, thereby providing an easy way to edit, visualize, analyze, monitor and control the search. Besides detecting remote homologies, Saturated BLAST can be used to maintain protein family databases and to search for new genes in genomic databases.

Algorithms↗
PubMed2000

Comparison of sequence profiles. Strategies for structural predictions using sequence information.

Distant homologies between proteins are often discovered only after three-dimensional structures of both proteins are solved. The sequence divergence for such proteins can be so large that simple comparison of their sequences fails to identify any similarity. New generation of sensitive alignment tools use averaged sequences of entire homologous families (profiles) to detect such homologies. Several algorithms, including the newest generation of BLAST algorithms and BASIC, an algorithm used in our group to assign fold predictions for proteins from several genomes, are compared to each other on the large set of structurally similar proteins with little sequence similarity. Proteins in the benchmark are classified according to the level of their similarity, which allows us to demonstrate that most of the improvement of the new algorithms is achieved for proteins with strong functional similarities, with almost no progress in recognizing distant fold similarities. It is also shown that details of profile calculation strongly influence its sensitivity in recognizing distant homologies. The most important choice is how to include information from diverging members of the family, avoiding generating false predictions, while accounting for entire sequence divergence within a family. PSI-BLAST takes a conservative approach, deriving a profile from core members of the family, providing a solid improvement without almost any false predictions. BASIC strives for better sensitivity by increasing the weight of divergent family members and paying the price in lower reliability. A new FFAS algorithm introduced here uses a new procedure for profile generation that takes into account all the relations within the family and matches BASIC sensitivity with PSI-BLAST like reliability.

Algorithms↗