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Adam Godzik

Publications and source records attributed to Adam Godzik.

At least 55 records · Page 3Linked to original sources
PubMed2003

Homotypic secretory vesicle fusion induced by the protein tyrosine phosphatase MEG2 depends on polyphosphoinositides in T cells.

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P(3) binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P(3) synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P(3) in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed2003

Characterization of the anti-apoptotic mechanism of Bcl-B.

Bcl-B protein is an anti-apoptotic member of the Bcl-2 family protein that contains all the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal transmembrane domain. Our previous results showed that Bcl-B binds Bax and suppresses apoptosis induced by over-expression of Bax; however, Bcl-B does not bind or suppress Bak. To explore the molecular basis for the differential binding and suppression of Bax and Bak, we studied the BH3 dimerization domains of Bax and Bak. Chimeric mutants of Bax and Bak were generated that swapped the BH3 domains of these pro-apoptotic proteins. Bcl-B associated with and blocked apoptosis induced by mutant Bak containing the BH3 domain of Bax, but not mutant Bax containing the BH3 domain of Bak. In contrast, Bcl-X(L) protein bound and suppressed apoptosis induction by Bax, Bak and both BH3-domain chimeras. A strong correlation between binding and apoptosis suppression was also obtained using a series of alanine substitutions spanning the length of the Bax BH3 domain to identify critical residues for Bcl-B binding. Conversely, using structure-based modelling to design mutations in the BH3-binding pocket of Bcl-B, we produced two Bcl-B mutants (Leu86-->Ala and Arg96-->Gln) that failed to bind Bax and that also were unable to suppress apoptosis induced by Bax over-expression. In contrast, other Bcl-B mutants that still bound Bax retained protective activity against Bax-induced cell death, thus serving as a control. We conclude that, in contrast with some other anti-apoptotic Bcl-2-family proteins, a strong correlation exists for Bcl-B between binding to pro-apoptotic multidomain Bcl-2 family proteins and functional apoptosis suppression.

Amino Acid Sequence↗
PubMed2003

Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization.

BACKGROUND: The correct folding and dimerization of tubulins, before their addition to the microtubular structure, needs a group of conserved proteins called cofactors A to E. The biochemical analysis of cofactors gave an insight to their general functions, however not much is known about the domain structure and detailed, molecular function of these proteins. RESULTS: Combining modelling and fold prediction tools, we present 3D models of all cofactors, including several previously unannotated domains of cofactors B-E. Apart from the new HEAT and Armadillo domains in cofactor D and an unusual spectrin-like domain in cofactor C, we have identified a new subfamily of ubiquitin-like domains in cofactors B and E. Together, these observations provide a reliable, molecular level model of cofactor complex. CONCLUSION: Distant homology searches allowed the identification of unknown regions of cofactors as self-reliant domains and allow us to present a detailed hypothesis of how a cofactor complex performs its function.

Amino Acid Sequence↗
PubMed2003

The PAAD/PYRIN-only protein POP1/ASC2 is a modulator of ASC-mediated nuclear-factor-kappa B and pro-caspase-1 regulation.

Proteins containing PAAD [pyrin, AIM (absent-in-melanoma), ASC [apoptosis-associated speck-like protein containing a CARD (caspase-recruitment domain)] and DD (death domain)-like] (PYRIN, DAPIN) domains are involved in innate immunity, regulating pathways leading to nuclear-factor-kappa B (NF-kappa B) and pro-caspase-1 activation. Many PAAD-family proteins have structures reminiscent of Nod-1, a putative intracellular sensor of lipopolysaccharide. Hereditary mutations in some of the PAAD-family genes are associated with auto-inflammatory diseases. Several of these proteins utilize the bipartite PAAD- and CARD-containing adapter protein ASC/TMS-1 (target of methylation-induced silencing) for linking to downstream signalling pathways. In the present paper, we describe characterization of human PAAD-only protein-1 (POP1)/ASC2, which is highly homologous with the PAAD domain of ASC, and which probably originated by gene duplication on chromosome 16. We demonstrate that POP1/ASC2 associates with ASC via PAAD-PAAD interactions and modulates NF-kappa B and pro-caspase-1 regulation by this adapter protein. In gene transfer experiments, POP1/ASC2 suppressed cytokine-mediated NF-kappa B activation similar to other PAAD-family proteins previously tested. Immunohistochemical studies showed expression of POP1/ASC2 predominantly in macrophages and granulocytes. We propose that POP1/ASC2 functions as a modulator of multidomain PAAD-containing proteins involved in NF-kappa B and pro-caspase-1 activation and innate immunity.

Amino Acid Sequence↗
PubMed2003

CARD6 is a modulator of NF-kappa B activation by Nod1- and Cardiak-mediated pathways.

We cloned a novel cDNA derived from the CARD6 gene locus on chromosome 5p12 of 311 amino acids in length. By immunoprecipitation we detected specific binding of this CARD6-encoding protein to Nod1 (CARD4), Cardiak (Rip2/Rick), NAC (NALP1/DEFCAP/CARD7), and TUCAN (CARD8/Cardinal/NDPP/Dakar), caspase recruitment domain (CARD)-containing proteins implicated in NF-kappa B and caspase-1 activation but not to other CARD family proteins. Cardiak and Nod1 (but not other CARD proteins) also exhibited opposing effects on CARD6 protein phosphorylation and expression, providing further evidence of functional interactions among these proteins in cells. In transfection experiments, the CARD6 protein suppressed NF-kappa B induction by Nod1 or Cardiak but did not interfere with NF-kappa B activation by the CARD-containing adapter protein Bcl10 or the cytokine tumor necrosis factor-alpha, demonstrating specificity of CARD6 for Nod-1 and Cardiak-dependent pathways. In contrast to its effects on Nod1- and Cardiak-dependent NF-kappa B activation, CARD6 did not interfere with caspase-1-dependent interleukin-1 beta secretion induced by Cardiak or Nod1. CARD6 also did not affect caspase activation and apoptosis induced by overexpression of Fas, Bax, or other pro-apoptotic stimuli. Thus, CARD6 represents a selective modulator of NF-kappa B activation by Cardiak and Nod1, adding to the repertoire of CARD-family proteins implicated in inflammatory responses and innate immunity.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A segment alignment approach to protein comparison.

MOTIVATION: Local structure segments (LSSs) are small structural units shared by unrelated proteins. They are extensively used in protein structure comparison, and predicted LSSs (PLSSs) are used very successfully in ab initio folding simulations. However, predicted or real LSSs are rarely exploited by protein sequence comparison programs that are based on position-by-position alignments. RESULTS: We developed a SEgment Alignment algorithm (SEA) to compare proteins described as a collection of predicted local structure segments (PLSSs), which is equivalent to an unweighted graph (network). Any specific structure, real or predicted corresponds to a specific path in this network. SEA then uses a network matching approach to find two most similar paths in networks representing two proteins. SEA explores the uncertainty and diversity of predicted local structure information to search for a globally optimal solution. It simultaneously solves two related problems: the alignment of two proteins and the local structure prediction for each of them. On a benchmark of protein pairs with low sequence similarity, we show that application of the SEA algorithm improves alignment quality as compared to FFAS profile-profile alignment, and in some cases SEA alignments can match the structural alignments, a feat previously impossible for any sequence based alignment methods.

Algorithms↗
PubMed2003

XOL-1, primary determinant of sexual fate in C. elegans, is a GHMP kinase family member and a structural prototype for a class of developmental regulators.

In Caenorhabditis elegans, an X chromosome-counting mechanism specifies sexual fate. Specific genes termed X-signal elements, which are present on the X chromosome, act in a concerted dose-dependent fashion to regulate levels of the developmental switch gene xol-1. In turn, xol-1 levels determine sexual fate and the activation state of the dosage compensation mechanism. The crystal structure of the XOL-1 protein at 1.55 A resolution unexpectedly reveals that xol-1 encodes a GHMP kinase family member, despite sequence identity of 10% or less. Because GHMP kinases, thus far, have only been characterized as small molecule kinases involved in metabolic pathways, for example, amino acid and cholesterol synthesis, XOL-1 is the first member that controls nonmetabolic processes. Biochemical investigations demonstrated that XOL-1 does not bind ATP under standard conditions, suggesting that XOL-1 acts by a mechanism distinct from that of other GHMP kinases. In addition, we have cloned a XOL-1 ortholog from Caenorhabditis briggsae, a related nematode that diverged from C. elegans approximately 50-100 million years ago. These findings demonstrate an unanticipated role for GHMP kinase family members as mediators of sexual differentiation and dosage compensation and, possibly, other aspects of differentiation and development.

Adenosine Triphosphatases↗
PubMed2003

The retinitis pigmentosa GTPase regulator (RPGR)- interacting protein: subserving RPGR function and participating in disk morphogenesis.

Retinitis pigmentosa is a photoreceptor degenerative disease leading to blindness in adulthood. Leber congenital amaurosis (LCA) describes a more severe condition with visual deficit in early childhood. Defects in the retinitis pigmentosa GTPase regulator (RPGR) and an RPGR-interacting protein (RPGRIP) are known causes of retinitis pigmentosa and LCA, respectively. Both proteins localize in the photoreceptor connecting cilium (CC), a thin bridge linking the cell body and the light-sensing outer segment. We show that RPGR is absent in the CC of photoreceptors lacking RPGRIP, but not vice versa. Mice lacking RPGRIP elaborate grossly oversized outer segment disks resembling a cytochalasin D-induced defect and have a more severe disease than mice lacking RPGR. Mice lacking both proteins are phenotypically indistinguishable from mice lacking RPGRIP alone. In vitro, RPGRIP forms homodimer and elongated filaments via interactions involving its coiled-coil and C-terminal domains. We conclude that RPGRIP is a stable polymer in the CC where it tethers RPGR and that RPGR depends on RPGRIP for subcellular localization and normal function. Our data suggest that RPGRIP is also required for disk morphogenesis, putatively by regulating actin cytoskeleton dynamics. The latter hypothesis may be consistent with a distant homology between the C-terminal domain of RPGRIP and an actin-fragmin kinase, predicted by fold recognition algorithms. A defect in RPGRIP encompasses loss of both functions, hence the more severe clinical manifestation as LCA.

Amino Acid Sequence↗
PubMed2003

A residue in the S2 subsite controls substrate selectivity of matrix metalloproteinase-2 and matrix metalloproteinase-9.

Matrix metalloproteinase (MMP)-2 and MMP-9 are closely related metalloproteinases that are implicated in angiogenesis. The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for understanding the structural basis of substrate recognition by the MMP family. Although the two MMPs exhibit some overlap in substrate recognition, our recent work showed that MMP-2 can cleave a set of peptide substrates that are only poorly recognized by MMP-9 (Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P(2) position of these peptide substrates dramatically reduced their selectivity for MMP-2. Inspection of the corresponding S(2) pocket of the substrate-binding cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2 contains Glu. Here, we test the hypothesis that this conservative substitution has a role in substrate selectivity. Mutation of Glu(412) in MMP-2 to Asp significantly reduced the hydrolysis of selective substrates, with only a minor effect on hydrolysis of non-selective substrates. The predominant effect of the mutation is at the level of k(cat), or turnover rate, with reductions reaching as high as 37-fold. The residues that occupy this position in other MMPs are highly variable, providing a potential structural basis for substrate recognition across the MMP family.

Amino Acid Substitution↗
PubMed2003

Fold recognition methods.

We are still missing a basic understanding of sequence/structure/function relationships in proteins. Analogy-based prediction algorithms remain the only reliable fold prediction tools. New methods, such as threading and hybrid threading/sequence fold recognition, can often recognize even the most distant homologues and, in some cases, even unrelated proteins with similar overall structures. This knowledge pushed the envelope of analogy-based function analysis to the point that the majority of newly sequenced genomes can be tentatively assigned to already characterized protein superfamilies. However, at this evolutionary distance, fold prediction is no longer equivalent to function prediction. Instead of having the same exact function, distantly related proteins might share some functional analogy that is not obvious to the casual observer. The main challenge facing the fold recognition field is to develop tools to follow the structure prediction with function prediction and analysis.

Algorithms↗
PubMed2003

Mitochondrial fission in apoptosis, neurodegeneration and aging.

A decline in mitochondrial function is well recognized in neurodegenerative diseases and aging, and is thought to play a causal role in their biology. Unfortunately, the molecular basis underlying this detrimental loss in mitochondrial function remains mysterious. Interestingly, mitochondria undergo frequent fission and fusion. This process is regulated by molecular machinery that has been highly conserved during evolution, including dynamin-related GTPases that manifest opposing effects. A balance between mitochondrial fission and fusion events is required for normal mitochondrial and cellular function. Emerging evidence indicates that mitochondria undergo rapid and extensive fission at an early stage during apoptosis. A clue that these new findings are of significance for the pathogenesis of neurodegenerative disease is provided by the observation that OPA-1, a dynamin-related GTPase regulating mitochondrial fusion, is mutated in humans with dominant optic atrophy, which is characterized by degeneration of retinal ganglion cells and childhood blindness. Loss of function of OPA-1, analogous to deficiency of its yeast homologue, Mgm1p, is expected to lead to mitochondrial fission, loss of mitochondrial DNA, respiratory deficits and an increase in reactive oxygen species. Here we review the molecular mediators controlling mitochondrial fission and fusion, and how death effector molecules may hijack this ancient machinery. A shift in the rate of mitochondrial fission or fusion may provide a new mechanistic explanation for the mitochondrial dysfunction in neurodegenerative diseases and normal aging, and may offer a new target for therapeutic intervention.

Aging↗
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↗