Carlos H.I. Ramos

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils IMPLICATIONS FOR CANCER

Background: p53 function is lost in more than 50% of tumors. Results: p53 aggregates into amyloid oligomers and fibrils in vitro and in breast cancer tissues; mutant p53 seeds amyloid aggregation of WT p53, a behavior typical of a prion. Conclusion: Prion-like aggregation is crucial for the negative dominance of mutant p53. Significance: The inhibition of aggregation could be a target for cancer therapy. Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.

The HD‐GYP domain of RpfG mediates a direct linkage between the Rpf quorum‐sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri

Bacteria use extracellular levels of small diffusible autoinducers to estimate local cell‐density (quorum‐sensing) and to regulate complex physiological processes. The quorum‐sensing signal transduction pathway of Xanthomonas spp. phytopathogens has special features that distinguish it from that of other pathogens. This pathway consists of RpfF, necessary for the production of the unique autoinducer ‘diffusible signalling factor’ (DSF), and RpfC and RpfG, a two‐component system necessary for the DSF‐dependent production of extracellular pathogenicity factors and cellular dispersion. Yeast two‐hybrid and direct in vitro assays were used to identify interactions involving the Rpf group of proteins. We show that RpfC, a protein consisting of N‐terminal transmembrane, histidine kinase, response‐regulator and C‐terminal histidine phosphotransfer domains interacts with both RpfG, a protein consisting of an N‐terminal response regulator domain and a C‐terminal HD‐GYP domain, and with RpfF. We also show that RpfC interacts with the only known homologue of ‘conditioned medium factor’, which is involved in quorum‐sensing in Dictyostelium discoideum under conditions of nutritional stress. Furthermore, RpfCG is shown to interact with a second two‐component system made up of NtrB and NtrC homologues. Finally we show that the recently characterized HD‐GYP phosphodiesterase domain of RpfG interacts directly with diguanylate cyclase GGDEF domain‐containing proteins coded by the Xanthomonas axonopodis pv. citri genome, which in other bacteria produce cyclic diGMP, an important second messenger involved in the regulation of complex bacterial processes including biofilm production, virulence and motility. These results demonstrate a direct physical linkage between quorum‐sensing and cyclic diGMP signalling pathways in bacteria.

Polypeptide transfer from Hsp40 to Hsp70 molecular chaperones

Heat shock protein 40 (Hsp40) co-chaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by Hsp40s and a common mechanism for polypeptide transfer to Hsp70.

The network interaction of the human cytosolic 90 kDa heat shock protein Hsp90: A target for cancer therapeutics.

In the cell, proteins interact within a network in which a small number of proteins are highly connected nodes or hubs. A disturbance in the hub proteins usually has a higher impact on the cell physiology than a disturbance in poorly connected nodes. In eukaryotes, the cytosolic Hsp90 is considered to be a hub protein as it interacts with molecular chaperones and co-chaperones, and has key regulatory proteins as clients, such as transcriptional factors, protein kinases and hormone receptors. The large number of Hsp90 partners suggests that Hsp90 is involved in very important functions, such as signaling, proteostasis and epigenetics. Some of these functions are dysregulated in cancer, making Hsp90 a potential target for therapeutics. The number of Hsp90 interactors appears to be so large that a precise answer to the question of how many proteins interact with this chaperone has no definitive answer yet, not even if the question refers to specific Hsp90s as one of the human cytosolic forms. Here we review the major chaperones and co-chaperones that interact with cytosolic Hsp90s, highlighting the latest findings regarding client proteins and the role that posttranslational modifications have on the function and interactions of these molecular chaperones. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Identification of New Protein-Protein Interactions Involving the Products of the Chromosome- and Plasmid-Encoded Type IV Secretion Loci of the Phytopathogen Xanthomonas axonopodis pv. citri

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.

Protein cutoff scanning: A comparative analysis of cutoff dependent and cutoff free methods for prospecting contacts in proteins

In this study, we carried out a comparative analysis between two classical methodologies to prospect residue contacts in proteins: the traditional cutoff dependent (CD) approach and cutoff free Delaunay tessellation (DT). In addition, two alternative coarse‐grained forms to represent residues were tested: using alpha carbon (CA) and side chain geometric center (GC). A database was built, comprising three top classes: all alpha, all beta, and alpha/beta. We found that the cutoff value at about 7.0 Å emerges as an important distance parameter. Up to 7.0 Å, CD and DT properties are unified, which implies that at this distance all contacts are complete and legitimate (not occluded). We also have shown that DT has an intrinsic missing edges problem when mapping the first layer of neighbors. In proteins, it may produce systematic errors affecting mainly the contact network in beta chains with CA. The almost‐Delaunay (AD) approach has been proposed to solve this DT problem. We found that even AD may not be an advantageous solution. As a consequence, in the strict range up to 7.0 Å, the CD approach revealed to be a simpler, more complete, and reliable technique than DT or AD. Finally, we have shown that coarse‐grained residue representations may introduce bias in the analysis of neighbors in cutoffs up to 6.8 Å, with CA favoring alpha proteins and GC favoring beta proteins. This provides an additional argument pointing to the value of 7.0 Å as an important lower bound cutoff to be used in contact analysis of proteins. Proteins 2009.

Low Resolution Structural Study of Two Human HSP40 Chaperones in Solution DJA1 FROM SUBFAMILY A AND DJB4 FROM SUBFAMILY B HAVE DIFFERENT QUATERNARY STRUCTURES

Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1–332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions.

New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri

We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.

Protein folding, misfolding and aggregation: evolving concepts and conformational diseases.

Proteins carry out many vital cellular functions determined by their precise 3-dimensional structures (the native conformations). Understanding how proteins fold has long been a major goal and can be of great therapeutic value. Failure to reach or maintain the correct folded structure can have serious consequences, as in the conformational diseases. The ultimate goal of folding studies is to predict structure from sequence, allowing the design of new functional proteins and prevention of aberrant disease-associated conformations.

Conserved central domains control the quaternary structure of Type I and Type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.