Biomedical subjects

Walid A Houry

Publications and source records attributed to Walid A Houry.

14 recordsLinked to original sources

PubMed2007

Molecular interaction network of the Hsp90 chaperone system.

Hsp90 is an essential and ubiquitous molecular chaperone that is required for the proper folding of a set of client proteins at a late stage in their folding process. In eukaryotes, cytoplasmic Hsp90 is absolutely essential for cell viability under all growth conditions. The functional cycle of the Hsp90 system requires a cohort of cochaperones and cofactors that regulate the activity of this chaperone. Hence, Hsp90 function is highly complex; in order to understand that complexity, several groups have attempted to map out the interaction network of this chaperone in yeast and mammalian systems using the latest available proteomic and genomic tools. Interaction networks emerging from these large scale efforts clearly demonstrate that Hsp90 plays a central role effecting multiple pathways and cellular processes. In yeast Saccharomyces cerevisiae, Hsp90 was shown to interact directly or indirectly with at least 10% of the yeast ORFs. The systematic application of large scale approaches to map out the Hsp90 chaperone network should allow the determination of the mechanisms employed by this chaperone system to maintain protein homeostasis in the cell.

HSP90 Heat-Shock Proteins↗

PubMed2006

Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX.

Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2-ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone.

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

MoxR AAA+ ATPases: a novel family of molecular chaperones?

The MoxR AAA+ family is a large, diverse group of ATPases that, so far, has been poorly studied. Members of this family are found throughout the Bacteria and Archaea superkingdoms, but have not yet been detected in Eukaryota. The limited experimental data available to date suggest that members of this family might have chaperone-like activities. Here we present an extensive phylogenetic analysis which builds upon our previously published work, and reveals that the MoxR family can be divided into at least seven subfamilies, including MoxR Proper (MRP), TM0930, RavA, CGN, APE2220, PA2707, and YehL. We also include a comprehensive overview and gene context analysis for each of these subfamilies. Our data reveal distinct conserved associations of certain MoxR family members with specific genes, including further support for our previously reported observation that many members of the MoxR AAA+ family are found near Von Willebrand Factor Type A (VWA) proteins and are likely to function with them. We propose, based on bioinformatic analyses and the available literature, that the MoxR AAA+ proteins function with VWA domain-containing proteins to form a chaperone system that is important for the folding/activation of proteins and protein complexes by primarily mediating the insertion of metal cofactors into the substrate molecules.

Adenosine Triphosphatases↗

PubMed2006

Hsp90 at the crossroads of genetics and epigenetics.

Hsp90 is a specialized molecular chaperone that is capable of buffering the expression of abnormal phenotypes. Inhibition of Hsp90 activity results in the expression of these phenotypes that are otherwise masked. Selection of offspring from the crossing of affected progenies results in inheritance and enrichment of these phenotypes, which can become independent of their original stimuli. The current combined evidence favours a model involving the interplay between genetics and epigenetics. The recent proteomics efforts to characterize the Hsp90 interaction networks provide further clues into the molecular mechanisms behind this complex phenomenon. This review summarizes the most recent experimental observations and briefly discusses the genetic and epigenetic views used in explaining the different observations.

Animals↗

PubMed2005

Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.

AAA+ ATPases are ubiquitous proteins that employ the energy obtained from ATP hydrolysis to remodel proteins, DNA, or RNA. The MoxR family of AAA+ proteins is widespread throughout bacteria and archaea but is largely uncharacterized. Limited work with specific members has suggested a potential role as molecular chaperones involved in the assembly of protein complexes. As part of an effort aimed at determining the function of novel AAA+ chaperones in Escherichia coli, we report the characterization of a representative member of the MoxR family, YieN, which we have renamed RavA (regulatory ATPase variant A). We show that the ravA gene exists on an operon with another gene encoding a protein, YieM, of unknown function containing a Von Willebrand Factor Type A domain. RavA expression is under the control of the sigmaS transcription factor, and its levels increase toward late log/early stationary phase, consistent with its possible role as a general stress-response protein. RavA functions as an ATPase and forms hexameric oligomers. Importantly, we demonstrate that RavA interacts strongly with inducible lysine decarboxylase (LdcI or CadA) forming a large cage-like structure consisting of two LdcI decamers linked by a maximum of five RavA oligomers. Surprisingly, the activity of LdcI does not appear to be affected by binding to RavA in a number of in vitro and in vivo assays, however, complex formation results in the stimulation of RavA ATPase activity. Data obtained suggest that the RavA-LdcI interaction may be important for the regulation of RavA activity against its targets.

Adenosine Triphosphatases↗

PubMed2005

Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release.

The highly conserved, 300-kDa cylindrical protease ClpP is an important component of the cellular protein quality machinery. It consists of 14 subunits arranged into two heptameric rings that enclose a large chamber containing the protease active sites. ClpP associates with ClpX and ClpA ATPases that unfold and translocate substrates into the protease catalytic chamber through axial pores located at both ends of the ClpP cylinder. Although the pathway of substrate delivery is well established, the pathway of product release is unknown. Here, we use recently developed transverse relaxation optimized spectroscopy (TROSY) of methyl groups to show that the interface between the heptameric rings exchanges between two structurally distinct conformations. The conformational exchange process has been quantified by magnetization exchange and methyl TROSY relaxation dispersion experiments recorded between 0.5 degrees C and 40 degrees C, so that the thermodynamic properties for the transition could be obtained. Restriction of the observed motional freedom in ClpP through the introduction of a cysteine linkage results in a protease where substrate release becomes significantly slowed relative to the rate observed in the reduced enzyme, suggesting that the observed motions lead to the formation of transient side pores that may play an important role in product release.

Electrophoresis, Polyacrylamide Gel↗

PubMed2005

Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.

Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.

Adenosine Triphosphatases↗

PubMed2005

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation.

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Hsp90: a chaperone for protein folding and gene regulation.

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.

Animals↗

PubMed2003

Solution structure of the dimeric zinc binding domain of the chaperone ClpX.

ClpX (423 amino acids), a member of the Clp/Hsp100 family of molecular chaperones and the protease, ClpP, comprise a multimeric complex supporting targeted protein degradation in Escherichia coli. The ClpX sequence consists of an NH2-terminal zinc binding domain (ZBD) and a COOH-terminal ATPase domain. Earlier, we have demonstrated that the zinc binding domain forms a constitutive dimer that is essential for the degradation of some ClpX substrates such as gammaO and MuA but is not required for the degradation of other substrates such as green fluorescent protein-SsrA. In this report, we present the NMR solution structure of the zinc binding domain dimer. The monomer fold reveals that ZBD is a member of the treble clef zinc finger family, a motif known to facilitate protein-ligand, protein-DNA, and protein-protein interactions. However, the dimeric ZBD structure is not related to any protein structure in the Protein Data Bank. A trimer-of-dimers model of ZBD is presented, which might reflect the closed state of the ClpX hexamer.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function.

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

Phylogenetic web profiler.

SUMMARY: Phylogenetic Web Profiler (PWP) is a web-based service designed to perform phylogenetic profiling of proteins against genomes. The current version offers a selection of 63 completed genomes and available plasmids as annotated in the PEDANT genome database. Unlike currently available applications, this tool offers several choices of ortholog prediction parameters including E-value cutoff, percent length difference tolerance, and annotation similarity. Additional features include tight integration with the PEDANT database and tools to analyze properties of predicted proteins. PWP should prove very useful for the analysis of functional-linkage between proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells.

The prevention of aberrant behavior of proteins is fundamental to cellular life. Protein homeostatic processes are present in cells to stabilize protein conformations, refold misfolded proteins, and degrade proteins that might be detrimental to the cell. Molecular chaperones and proteases perform a major role in these processes. In bacteria, the main cytoplasmic components involved in protein homeostasis include the chaperones trigger factor, DnaK/DnaJ/GrpE, GroEL/GroES, HtpG, as well as ClpB and the proteases ClpXP, ClpAP, HslUV, Lon, and FtsH. Based on recent genome sequencing efforts, it was surprising to find that the Mycoplasma, a genus proposed to include a minimal form of cellular life, do not contain certain major members of the protein homeostatic network, including GroEL/GroES. We propose that, in mycoplasmas, there has been a fundamental shift towards favoring processes that promote protein degradation rather than protein folding. The arguments are based on two different premises: (1) the regulation of stress response in Mycoplasma and (2) the unique characteristics of the Mycoplasma proteome.

Bacterial Proteins↗