Search PubMed⌕ Search

Biomedical subjects

Adam Godzik

Publications and source records attributed to Adam Godzik.

At least 37 records · Page 2Linked to original sources
PubMed2003

Flexible structure alignment by chaining aligned fragment pairs allowing twists.

MOTIVATION: Protein structures are flexible and undergo structural rearrangements as part of their function, and yet most existing protein structure comparison methods treat them as rigid bodies, which may lead to incorrect alignment. RESULTS: We have developed the Flexible structure AlignmenT by Chaining AFPs (Aligned Fragment Pairs) with Twists (FATCAT), a new method for structural alignment of proteins. The FATCAT approach simultaneously addresses the two major goals of flexible structure alignment; optimizing the alignment and minimizing the number of rigid-body movements (twists) around pivot points (hinges) introduced in the reference protein. In contrast, currently existing flexible structure alignment programs treat the hinge detection as a post-process of a standard rigid body alignment. We illustrate the advantages of the FATCAT approach by several examples of comparison between proteins known to adopt different conformations, where the FATCAT algorithm achieves more accurate structure alignments than current methods, while at the same time introducing fewer hinges.

Algorithms↗
PubMed2003

Comparative analysis of apoptosis and inflammation genes of mice and humans.

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFkappaB induction. Proteins involved in apoptosis and NFkappaB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFkappaB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.

Animals↗
PubMed2003

Homology modeling provides insights into the binding mode of the PAAD/DAPIN/pyrin domain, a fourth member of the CARD/DD/DED domain family.

The PAAD/DAPIN/pyrin domain is the fourth member of the death domain superfamily, but unlike other members of this family, it is involved not only in apoptosis but also in innate immunity and several other processes. We have identified 40 PAAD domain-containing proteins by extensively searching the genomes of higher eukaryotes and viruses. Phylogenetic analyses suggest that there are five categories of PAAD domains that correlate with the domain architecture of the entire proteins. Homology models built on CARD and DD structures identified functionally important residues by studying conservation patterns on the surface of the models. Surface maps of each subfamily show different distributions of these residues, suggesting that domains from different subfamilies do not interact with each other, forming independent regulatory networks. Helix3 of PAAD is predicted to be critical for dimerization. Multiple alignment analysis and modeling suggest that it may be partly disordered, following a new paradigm for interaction proteins that are stabilized by protein-protein interactions.

Amino Acid Sequence↗
PubMed2002

Tyrosine phosphorylation of VHR phosphatase by ZAP-70.

The ZAP-70 tyrosine kinase is a key component of the signaling machinery for the T cell antigen receptor (TCR). Whereas recruitment and activation of ZAP-70 are relatively well understood, the proteins phosphorylated by ZAP-70 are incompletely known. We report here that VHR, a Vaccinia virus VH1-related dual-specific protein phosphatase that inactivates the mitogen-activated kinases Erk2 and Jnk, is phosphorylated at Y138 by ZAP-70. Tyr138 phosphorylation was required for VHR to inhibit the Erk2-Elk-1 pathway and, conversely, the VHR(Y138F) mutant augmented TCR-induced Erk2 kinase and activation of the gene encoding interleukin 2. These results suggest that VHR is a target for ZAP-70 and tempers activation of the Erk2 pathway in a ZAP-70-controlled manner.

DNA-Binding Proteins↗
PubMed2002

Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline.

Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Cloning, Molecular↗
PubMed2002

A novel PAAD-containing protein that modulates NF-kappa B induction by cytokines tumor necrosis factor-alpha and interleukin-1beta.

PAAD domains are found in diverse proteins of unknown function and are structurally related to a superfamily of protein interaction modules that includes death domains, death effector domains, and Caspase activation and recruitment domains. Using bioinformatics strategies, cDNAs were identified that encode a novel protein of 110 kDa containing a PAAD domain followed by a putative nucleotide-binding (NACHT) domain and several leucine-rich repeat domains. This protein thus resembles Cryopyrin, a protein implicated in hereditary hyperinflammation syndromes, and was termed PAN2 for PAAD and NACHT-containing protein 2. When expressed in HEK293 cells, PAN2 suppressed NF-kappaB induction by the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), suggesting that this protein operates at a point of convergence in these two cytokine signaling pathways. This PAN2-mediated suppression of NF-kappaB was evident both in reporter gene assays that measured NF-kappaB transcriptional activity and electromobility shift assays that measured NF-kappaB DNA binding activity. PAN2 also suppressed NF-kappaB induction resulting from overexpression of several adapter proteins and protein kinases involved in the TNF or IL-1 receptor signal transduction, including TRAF2, TRAF6, RIP, IRAK2, and NF-kappaB-inducing kinase as well as the IkappaB kinases IKKalpha and IKKbeta. PAN2 also inhibited the cytokine-mediated activation of IKKalpha and IKKbeta as measured by in vitro kinase assays. Furthermore, PAN2 association with IKKalpha was demonstrated by co-immunoprecipitation assays, suggesting a direct effect on the IKK complex. These observations suggest a role for PAN2 in modulating NF-kappaB activity in cells, thus providing the insights into the potential functions of PAAD family proteins and their roles in controlling inflammatory responses.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases.

In our study, we characterized the substrate recognition properties of membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP-14), a key enzyme in tumor cell invasion and metastasis. A panel of optimal peptide substrates for MT1-MMP was identified using substrate phage display. The substrates can be segregated into four groups based on their degree of selectivity for MT1-MMP. Substrates with poor selectivity for MT1-MMP are comprised predominately of the Pro-X-X- downward arrow-X(Hy) motif that is recognized by a number of MMPs. Highly selective substrates lack the characteristic Pro at the P(3) position; instead they contain an Arg at the P(4) position. This P(4) Arg is essential for efficient hydrolysis and for selectivity for MT1-MMP. Molecular modeling indicates that the selective substrates adopt a linear conformation that extends along the entire catalytic pocket of MT1-MMP, whereas non-selective substrates are kinked at the conserved P(3) Pro residue. Importantly, the selective substrates can be made non-selective by insertion of a proline kink at P(3), without significantly reducing overall k(cat)/K(m) values. Altogether the study provides a structural basis for selective and non-selective substrate recognition by MT1-MMP. The findings in this report are likely to explain several aspects of MT1-MMP biology.

Catalytic Domain↗
PubMed2002

CADD, a Chlamydia protein that interacts with death receptors.

We report here the identification of a bacterial protein capable of interacting with mammalian death receptors in vitro and in vivo. The protein is encoded in the genome of Chlamydia trachomatis and has homologues in other Chlamydia species. This protein, which we refer to as "Chlamydia protein associating with death domains" (CADD), induces apoptosis in a variety of mammalian cell lines when expressed by transient gene transfection. Apoptosis induction can be blocked by Caspase inhibitors, indicating that CADD triggers cell death by engaging the host apoptotic machinery. CADD interacts with death domains of tumor necrosis factor (TNF) family receptors TNFR1, Fas, DR4, and DR5 but not with the respective downstream adaptors. In infected epithelial cells, CADD is expressed late in the infectious cycle of C. trachomatis and co-localizes with Fas in the proximity of the inclusion body. The results suggest a role for CADD modulating the apoptosis pathways of cells infected, revealing a new mechanism of host-pathogen interaction.

Amino Acid Sequence↗
PubMed2002

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

Animals↗
PubMed2002

Discovering new genes with advanced homology detection.

Most genome annotation protocols combine ab initio predictions with transcription and homology analyses to produce reliable gene predictions but they often fail to detect many actual genes. Alternative approaches involving more sensitive homology recognition methods are playing an increasingly important role in the next stage of gene discovery. The hunt for new genes is far from over.

Database Management Systems↗
PubMed2002

Cloning and characterization of glucose transporter 11, a novel sugar transporter that is alternatively spliced in various tissues.

We have cloned and characterized a novel glucose transporter (GLUT11) that is alternatively spliced. The GLUT11 gene maps to chromosome 22q11.2 and consists of 13 exons. The long form (GLUT11-L) cDNA uses 13 exons to produce a protein containing 503 amino acids. The short form of GLUT11 (GLUT-11) cDNA is missing exon 2 and produces a protein of 496 amino acids with a 14 amino acid N-terminal difference compared to the long form. GLUT11 has significant similarity to known GLUTs and contains 12 putative membrane-spanning helices along with sugar transporter signature motifs that have previously been shown to be essential for transport activity. The putative glycosylation site of GLUT11 is present in loop 1. Northern blot analysis showed that GLUT11 mRNA is expressed in a number of tissues and most abundantly in the skeletal muscle and heart. RT-PCR assay showed that GLUT11 is alternatively spliced and the two isoforms are distributed differently in various tissues. Immunofluorescence microscopy demonstrated that GLUT11-L resides on the plasma membrane when overexpressed in HEK293T cells. Western blot analysis revealed that GLUT11-L runs as a broad band of approximately 42 kDa that was converted to a 38 kDa polypeptide by PNGase F digestion. Furthermore, a liposome reconstitution functional assay showed that GLUT11-L has glucose transport activity.

Alternative Splicing↗
PubMed2002

Tolerating some redundancy significantly speeds up clustering of large protein databases.

MOTIVATION: Sequence clustering replaces groups of similar sequences in a database with single representatives. Clustering large protein databases like the NCBI Non-Redundant database (NR) using even the best currently available clustering algorithms is very time-consuming and only practical at relatively high sequence identity thresholds. Our previous program, CD-HI, clustered NR at 90% identity in approximately 1 h and at 75% identity in approximately 1 day on a 1 GHz Linux PC (Li et al., Bioinformatics, 17, 282, 2001); however even faster clustering speed is needed because the size of protein databases are rapidly growing and many applications desire a lower attainable thresholds. RESULTS: For our previous algorithm (CD-HI), we have employed short-word filters to speed up the clustering. In this paper, we show that tolerating some redundancy makes for more efficient use of these short-word filters and increases the program's speed 100 times. Our new program implements this technique and clusters NR at 70% identity within 2 h, and at 50% identity in approximately 5 days. Although some redundancy is present after clustering, our new program's results only differ from our previous program's by less than 0.4%.

Algorithms↗
PubMed2002

Sequence clustering strategies improve remote homology recognitions while reducing search times.

Sequence databases are rapidly growing, thereby increasing the coverage of protein sequence space, but this coverage is uneven because most sequencing efforts have concentrated on a small number of organisms. The resulting granularity of sequence space creates many problems for profile-based sequence comparison programs. In this paper, we suggest several strategies that address these problems, and at the same time speed up the searches for homologous proteins and improve the ability of profile methods to recognize distant homologies. One of our strategies combines database clustering, which removes highly redundant sequence, and a two-step PSI-BLAST (PDB-BLAST), which separates sequence spaces of profile composition and space of homology searching. The combination of these strategies improves distant homology recognitions by more than 100%, while using only 10% of the CPU time of the standard PSI-BLAST search. Another method, intermediate profile searches, allows for the exploration of additional search directions that are normally dominated by large protein sub-families within very diverse families. All methods are evaluated with a large fold-recognition benchmark.

Amino Acid Motifs↗
PubMed2002

In search for more accurate alignments in the twilight zone.

A major bottleneck in comparative modeling is the alignment quality; this is especially true for proteins whose distant relationships could be reliably recognized only by recent advances in fold recognition. The best algorithms excel in recognizing distant homologs but often produce incorrect alignments for over 50% of protein pairs in large fold-prediction benchmarks. The alignments obtained by sequence-sequence or sequence-structure matching algorithms differ significantly from the structural alignments. To study this problem, we developed a simplified method to explicitly enumerate all possible alignments for a pair of proteins. This allowed us to estimate the number of significantly different alignments for a given scoring method that score better than the structural alignment. Using several examples of distantly related proteins, we show that for standard sequence-sequence alignment methods, the number of significantly different alignments is usually large, often about 10(10) alternatives. This distance decreases when the alignment method is improved, but the number is still too large for the brute force enumeration approach. More effective strategies were needed, so we evaluated and compared two well-known approaches for searching the space of suboptimal alignments. We combined their best features and produced a hybrid method, which yielded alignments that surpassed the original alignments for about 50% of protein pairs with minimal computational effort.

Algorithms↗
PubMed2001

Identification and characterization of DEDD2, a death effector domain-containing protein.

A novel Death Effector Domain-containing protein was identified, DEDD2, which is closest in amino acid sequence homology to death effector domain-containing DNA-binding protein, DEDD. DEDD2 mRNA is expressed widely in adult human tissues with highest levels in liver, kidney, and peripheral blood leukocytes. DEDD2 interacts with FLIP, but not with Fas-associated death domain (FADD) or caspase-8. Overexpression of DEDD2 induces moderate apoptosis and results in substantial sensitization to apoptosis induced by Fas (CD95/APO-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), or FADD. In contrast, Bax- or staurosporine-mediated cell death is not affected by expression of DEDD2. Fluorescence microscopy showed that overexpressed DEDD2 translocates to the nucleus, which is dependent on the presence of a bipartite nuclear localization signal in the DEDD2 protein. Mutagenesis studies revealed that the translocation of the DED of DEDD2 to the nucleus is essential for its pro-apoptotic activity. These findings suggest that DEDD2 is involved in the regulation of nuclear events mediated by the extrinsic apoptosis pathway.

Active Transport, Cell Nucleus↗
PubMed2001

Inhibition of T cell antigen receptor signaling by VHR-related MKPX (VHX), a new dual specificity phosphatase related to VH1 related (VHR).

A cDNA encoding a novel, human, dual-specific protein phosphatase was identified in the Incyte data base. The open reading frame predicted a protein of 184 amino acids related to the Vaccinia virus VH1 and human VH1-related (VHR) phosphatases. Expression VHR-related MKPX (VHX) was highest in thymus, but also detectable in monocytes and lymphocytes. A VHX-specific antiserum detected a protein with an apparent molecular mass of 19 kDa in many cells, including T lymphocytes and monocytes. VHX expression was not induced by T cell activation, but decreased somewhat at later time points. In vitro, VHX dephosphorylated the Erk2 mitogen-activated protein kinase with faster kinetics than did VHR, which is thought to be specific for Erk1 and 2. When expressed in Jurkat T cells, VHX had the capacity to suppress T cell antigen receptor-induced activation of Erk2 and of an NFAT/AP-1 luciferase reporter, but not an NF-kappaB reporter. Thus, VHX is a new member of the VH1/VHR group of small dual-specific phosphatases that act in mitogen-activated protein kinase signaling pathways.

Amino Acid Sequence↗
PubMed2001

A unique substrate recognition profile for matrix metalloproteinase-2.

The catalytic domains of the matrix metalloproteinases (MMPs) are structurally homologous, raising questions as to the degree of distinction, or overlap, in substrate recognition. The primary objective of the present study was to define the substrate recognition profile of MMP-2, a protease that was historically referred to as gelatinase A. By cleaving a phage peptide library with recombinant MMP-2, four distinct sets of substrates were identified. The first set is structurally related to substrates previously reported for other MMPs. These substrates contain the PXX/X(Hy) consensus motif (where X(Hy) is a hydrophobic residue) and are not generally selective for MMP-2 over the other MMPs tested. Two other groups of substrates were selected from the phage library with similar frequency. Substrates in group II contain the L/IXX/X(Hy) consensus motif. Substrates in group III contain a consensus motif with a sequence of X(Hy)SX/L, and the fourth set of substrates contain the HXX/X(Hy) sequence. Substrates in Group II, III, and IV were found to be 8- to almost 200-fold more selective for MMP-2 over MMP-9. To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P(2) residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P(2) subsite as a primary determinant in substrate distinction.

Amino Acid Sequence↗