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Characterization of the role of HSP70 system and HSP22 as a disaggregate complex in human

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Supervisor abroad: Jason C. Young
Research place: McGill University (Canada)
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships abroad - Research Internship - Doctorate
Grant number:16/03764-0
Duration: August 01, 2016 - July 31, 2017
Abstract
The chaperones of the Hsp70 family have central role in the cellular network of molecular chaperones. These chaperones are engaged in many biological processes, including the folding of recently synthesized proteins to the native state, prevention of aggregation, solubilization and refolding of misfolded or aggregated proteins and transport of proteins across membranes. Hsp70 generally depend on co-chaperones to regulate ATPase cycles and affinity for substrate, especially the J-domain proteins (Hsp40) and Hsp110, nucleotide exchange factors (NEF). Although the ATPase cycle of Hsp70 proteins is well known, structural information on the larger complexes formed by Hsp70, Hsp40 and client protein is still limited because of its transient nature. Apart from the lack of structural information about Hsp70 complexes, functional information is also scarce. Intracellular accumulation of protein aggregates is an important mark of stressed cells and can be a serious threat to the health of organisms, generally culminating in diverse neuro-degenerative disorders, such as Huntington's, Alzheimer's, Parkinson's and Amyotrophic Lateral Sclerosis, which have no known cure yet. Despite metazoan cells lack the main disaggregase chaperone Hsp100, they still are able to eliminate protein aggregates. This issue was unresolved but, recently, protein disaggregation experiments using purified human and C. elegans components showed that interaction between class A and B J-proteins and Hsp70 with the NEF Hsp110 is the responsible for reactivate client aggregated after thermal stress. Specifically, class B DNAJB1 cooperated with class A DNAJA2 (or DNAJA1). In these findings, the disaggregase activity of Hsp70 system depends on the presence of a sHsp from yeast (Hsp26), however the mechanism by which these sHsps play a role in the process still needs to be studied in detail. Because of the importance of this system in protein quality control biology, the goal of this proposal is to investigate the disaggregase system in human molecular chaperones. For this, the objectives are: (1) establish a protocol for purification of human HspB8/Hsp22 and characterize its role in the disaggregation process in vitro; (2) investigate the role of Hsp22, as a potential partner of Hsp70/J-proteins/Hsp110, in disaggregation processes by performing experiments in cultured cells; (3) characterize the effect of J-proteins from class A (DJA4) and B (DJB4) on the activity of human Hsp70. (AU)

Deciphering structural information of Hsp90 chaperonecomplexed with hop and chip co-chaperones using highresolution mass spectrometry and chemical cross-link

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Supervisor abroad: Manfredo Riccardo Quadroni
Research place: Université de Lausanne (UNIL) (Switzerland)
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships abroad - Research Internship - Post-doctor
Grant number:15/21930-1
Duration: November 20, 2015 - December 19, 2015
Abstract
The Hsp90 (heat shock protein 90 kDa) plays a key role in the maturation of at least 10% of all proteins produced by the cell, which makes it important for many cellular processes such signaling, proteostasis, epigenetics, telomere maintenance, innate immunity, etc. Obviously, changes in this 'highly connected node' leads to disturbances in cell function resulting in drastic consequences for the organism. Hsp90 appears to play a central role in the pathology of several types of cancer as many kinases are clients and are involved in the development of cancer. As a matter of fact, specific inhibitors of Hsp90 and co-chaperones are under investigation in clinical trials. No human cytosolic Hsp90 had its full three-dimensional structure determined and the mechanisms by which this chaperone functions and is modulated are not yet fully known. Even the set of co-chaperones that play key role in the processes aforementioned, are not yet completly understood. The obvious therapeutic potential that Hsp90 from human, and Hsp90 from other organisms, have, make essential to unravel the forces that stabilize their structure and the mechanisms by which their functions are modulated resulting in the maturation of proteins clients. The group of Pr. Carlos Ramos have already investigated the conformational and functional aspect of Hsp90, but as mentioned before there is still the impact ofits interaction with its co-chaperonessuch as HOP, CHIP or SGT1,to elucidate. To reach this goal we propose to analyze chemically cross-linked chaperone/co-chaperones complexes using fast MS/MS scan coupled with high resolutionmass spectrometry in order to: 1) Determine the conformational aspects of Hsp90 complexed with co-chaperones, 2) Determine the conformational aspects of co-chaperones alone and complexed with Hsp90, 3) Investigate the interaction of Hsp90 with several co-chaperones using different type of cross-link (zero length cross-link and 7-11 Å cross-link). Those analyzes will be possible grace of the contribution of Dr. ManfredoQuadronigroup who possess the state-of-the-art in Mass Spectrometryarea. The objective during one month is to pre-fractionate cross-linked samples, inject them in mass spectrometry and analyze those data. (AU)

Functional characterization of R2TP-Hsp90 complex in different organisms

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Supervisor abroad: Walid A. Houry
Research place: University of Toronto (Canada)
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships abroad - Research Internship - Doctorate
Grant number:15/13521-4
Duration: October 15, 2015 - March 30, 2016
Abstract
The maintenance of protein homeostasis is critical for cellular survival, and molecular chaperones are essential for this process. One of the more important proteins involved in this process is Hsp90, which also has key roles in a plethora of cellular functions. Dr. Walid A. Houry's group at University of Toronto (Canada) identified 627 putative Hsp90 interacting proteins and discovered the R2TP complex in yeast. R2TP consists of the highly conserved in eukaryotes called Rvb1 and Rvb2 helicases which bind tightly Pih1 and Tah1, the Hsp90 cofactors. Notably, many studies have revealed that a large range of carcinomas exhibit increased expression of Rvb1 and Rvb2 proteins. Furthermore, yeast Pih1, known as PIH1D1 in humans, interacts with various components in the cell and Tah1 contains tetratricopeptide repeat (TPR) motifs, which interact with the MEEVD motif on Hsp90 C-terminus that is known to mediate the interaction of Hsp90 with its co-chaperones. In this sense, Dr. Carlos Ramos's group at Unicamp (Brazil) has been studying orthologs of the R2TP complex from Leishmania major, an obligate intracellular protozoan parasite which causes a human disease named cutaneous leishmaniasis. Therefore, the R2TP complex appears to play a critical role in the rapid multiplication of these parasites and our group speculates that the R2TP complex is also important for the parasite Leishmania major as in yeast and humans. Despite the advances in studies of the R2TP-Hsp90 complex in different biological processes, the understanding of its cellular roles and specifically mechanism of function is still poorly understood. Therefore, this project will undertake a collaborative effort of both groups aiming at elucidating the molecular basis of R2TP activity in different organisms. Considering the above, this project aims to further investigate R2TP complex by (1) characterizing the ATPase and helicase activity of Rvb1/2 and (2) use the yeast and mammalian cells system to understand the molecular basis of R2TP complex. The knowledge gained with this study will improve the experimental effort in understand the protozoan ortholog. Lastly, given the techniques and approaches wich a phD student will use, this project will provide a unique opportunity to expand her scientific horizons to 'think outside the box' and tackle a problem from different angles. (AU)

Characterization of the role of ATPase domains and human co-chaperones Hsp40 and hop in cellular mechanisms of thermal tolerance and suppression of protein aggregation

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Doctorate
Grant number:14/25967-4
Duration: March 01, 2015 - February 28, 2018
Cooperation agreement with FAPESP: Coordination of Improvement of Higher Education Personnel (CAPES)
Abstract
A number of human diseases known as 'conformational' is related to the incorrect protein folding. Misfolded proteins do not function correctly and may also form aggregates or amyloids, which are associated with hundreds of diseases including Alzheimer and Parkinson. Molecular chaperones and Hsps (heat shock proteins) participate in a cellular system known as PQC (Protein Quality Control) involved in maintaining protein homeostasis. There are specialized chaperones in aiding protein folding ('foldases'), in the inhibition of the aggregate formation ('holders') and in the resolubilisation of aggregates ('desagregases'). Interestingly, specialized desagregases have not yet been found in metazoan cell cytosol suggesting that other proteins may be involved with this function. Our group has extensive background in the study of protein folding and aggregation of proteins and chaperones and in this project we seek detailed information about the cellular mechanisms of protection against thermal stress and protein aggregation. Therefore, we will use yeast as a model to test thermotolerance and aggregation of prions and amyloid. First, a chimera of the ATPase domain of a human protein involved in neuronal diseases and suggested as a chaperone role, and Hsp104, yeast desagregase, will be produced and studied. We will also study the role of human co-chaperones Hsp40 and Hop in the suppression of protein aggregation since their yeast counterparts have this role. Another objective of which depends on the results of the above objectives, is the investigation of whether these proteins interact to form a protein complex that participates in the PQC system. (AU)

Study of important chaperone-interaction from the celular Protein Quality Control system in higher eukaryotes

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Doctorate
Grant number:14/00076-0
Duration: April 01, 2014 - March 31, 2017
Associated research grant:12/50161-8 - Study of the structure and function of the Hsp90 chaperone with emphasis on its role in cellular homeostasis, AP.TEM
Abstract
Proteins are responsible by a huge number of functions in cells. This functions are directly related to the proteins fold structure. The proteins folding may not occur naturally in cells, so there is a specialized system, known Protein Quality Control (PQC). The PQC is formed by the sub-system of chaperones, which facilitates the correct folding of proteins, and the sub-system proteosome, responsible for degradation. The proteins of PQC system are more express in conditions of stress, when the wrong folding is easier. The main objective of our group is understand how the PQC system works in eukariotes, specially in human and plants. This project has the objetive of investigate the interactions among chaperones and Hsps considered relevant for the function of the PQC system. Thus, the specifics objectives are: 1) Cloning, express, purify and characterize the co-chaperone CHIP in a plant. This protein has a bind domain to Hsp70 and Hsp90 chaperones and other domain that interacts with E3 ubiquitin-ligase, so this protein is an important link between the two sub-system of the PQC. 2) Investigate the mechanisms of interaction between Hsp100 and smHsps in plants. 3) Compare the conformation of four Hsp40 human chaperones and chacterize its interactions with unfold proteins and Hsp70 (AU)

Studies of proteins in the eukaryotic Hsp90 proteomic hub: functional characterization of the human and Leishmania braziliensis p23 co-chaperones and a possible metazoan hsp100/clpB ortholog utilizing yeast as a surrogate expression system

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Post-Doctorate
Grant number:13/11500-4
Duration: September 01, 2013 - August 31, 2014
Associated research grant:12/50161-8 - Study of the structure and function of the Hsp90 chaperone with emphasis on its role in cellular homeostasis, AP.TEM
Abstract
The maintenance of protein homeostasis is critical for cellular survival, and molecular chaperones are essential for this process. One important protein for facilitating refolding is Hsp90, which also has key roles in a host of other cellular functions, such as proteostasis, epigenetics, telomere maintenance, innate immunity, and biological signaling, among others. Hence, disruption of Hsp90 function can have dramatic, if not lethal, consequences on the organism. One example of this includes the fact that several types of cancer are dependent on upregulated Hsp90 activity, making it an important drug target. Furthermore, interfering with Hsp90 activity has recently become a goal in the design of anti-parasitic agents. Interestingly, some protozoans, such as Leishmania braziliensis, contain two forms of p23, an Hsp90 co-chaperone and modulator of Hsp90 function. Likewise, the human ortholog of p23 can undergo caspase cleavage to P18, which has a completely different function than P23, and has been implicated in apoptosis. Lastly, some Hsp90 co-chaperones have been found to interact with Hsp104, and we propose that this could be conserved in higher eukaryotes. This project aims to further investigate Hsp90 interactions by 1) characterizing human and Leishmania braziliensis p23 protein variants using yeast as a surrogate expression system to measure in vivo activity, 2) further studies to establish an Hsp104-like protein activity in metazoans (ClpB homolog) by determining how its heterologous expression in yeast effects a panel of insoluble aggregate forming proteins, and 3) using yeast as an expression system to purify the human ClpB homolog in order to perform in vitro biochemical activity assays. These studies will significantly contribute to the current knowledge of human chaperone function, and are relevant to the parent project theme of Hsp90. (AU)

Characterization of human genes as potential chaperone with desagregase function

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Doctorate
Grant number:13/10939-2
Duration: August 01, 2013 - January 16, 2017
Associated research grant:12/50161-8 - Study of the structure and function of the Hsp90 chaperone with emphasis on its role in cellular homeostasis, AP.TEM
Abstract
A growing number of diseases have been correlated with the incorrect folding and protein aggregation. To protect the cells of these factors there are a group of proteins known as molecular chaperones that are intrinsically involved in protein homeostasis. These proteins help the protein folding and participate in many other cellular processes such as translocation and signaling. The most important of these chaperone functions is the capacity to disaggregate amyloid proteins. This chaperone belongs to the Hsp100 family and is called disaggregase. This chaperone is present in prokaryotes, some plants, parasites, but curiously is missing in animals. This project aims at understanding the mechanism of a human protein, now known as 'human ClpB homolog' that in recent results from our group showed function and complementation of yeast that had the disaggregase gene deleted, and to definitely establish this as a molecular chaperone disaggregase. The potential of disaggregases action in the protein aggregates can suggest their use in therapies against disease-associated conformations such as Alzheimer's and Parkinson's. The techniques to be used to achieve these results are: evaluation of expression levels in HeLa cells under conditions of stress, silencing expression 'RNAi knock-down'; heterologous expression in yeast his-tag for purification with co-interactors, and to establish a purification method of the soluble protein for structural and functional studies in vitro. Moreover, there is a possibility that the animals could have several different disaggregases with 'low activity' than a single disaggregase with 'high activity', another aim of this proposal is to test one or more candidate genes. Then, the achievement of this project has a great deal to contribute to our understanding the action mechanisms of this protein in humans, because the literature currently offers only three articles that report the function of this protein in metazoan. (AU)

Expression and characterization of two recombinant manganese-containing catalases (MnCats) from Bacillus pumilus SAFR-032: structural and functional studies, and analysis of binding affinity of metal divalent cations

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Post-Doctorate
Grant number:12/05441-2
Duration: October 01, 2012 - January 31, 2014
Abstract
Excessive hydrogen peroxide (H2O2) is harmful for almost all cell components, and its rapid and efficient removal is of essential importance for aerobic organisms. H2O2 is degraded by catalases, an enzyme able to dismutate the H2O2 to water and molecular oxygen. Nature has evolved three protein families that are able to catalyze this process. Two of these families are heme enzymes comprising the most abundant group found in all kingdoms of life. The third group is a minor bacterial protein family with a dimanganese active site called manganese catalases (MnCats) widely distributed over microbial community, being the oldest H2O2 dismutating catalyst enzymes. Actually, 100 MnCats genes have been annotated in genomic databases, although the assignment of many of these proteins needs to be experimentally verified. Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, g-radiation, H2O2, desiccation and chemical disinfection, and B. pumilus SAFR-032, isolated from the Jet Propulsion Lab spacecraft assembly facility (JPL/NASA), survives to these conditions to the extreme. Analysis of the B. pumilus SAFR-032 genome reveals two putative MnCats genes (BPUM_2346 and BPUM_1305), that may have important properties that contribute to the extreme H2O2 resistance of this organism. Our proposal is to express, characterize structurally and functionally, and analyse the binding affinity of different divalent metal cations to these metalloproteins. Additionally, with the expected results from this proposal, we aim to understand the relations between function and structure of MnCats, which may help to design specific inhibitors to prevent diseases in humans, animals and plants related to these enzymes. (AU)

Study of the structure and function of the Hsp90 chaperone with emphasis on its role in cellular homeostasis

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Co-Principal Investigators:

Maria Isabel Nogueira Cano ; Julio Cesar Borges

Field of knowledge:Biological Sciences - Biochemistry
Support type:Research Projects - Thematic Grants
Grant number:12/50161-8
Duration: August 01, 2012 - July 31, 2017
Abstract
The Hsp90 chaperone (heat shock protein 90 kDa) is found in all organisms and in all cellular compartments, and the two types found in the eukaryotic cytosol interact with at least 10% of all proteins produced by the cell. The Hsp90 plays a key role in the maturation of these proteins, which makes it important for many cellular processes such signaling, proteostasis, epigenetics, telomere maintenance, innate immunity, etc. Obviously, changes in this 'highly connected node', as one can label Hsp90, leads to disturbances in cell function resulting in drastic consequences for the organism. For example, Hsp90 appears to play a central role in the pathology of several types of cancer as many kinases are clients and are involved in the development of cancer. As a matter of fact, specific inhibitors of Hsp90 and co-chaperones are under investigation in clinical trials. No human cytosolic Hsp90 had its full three-dimensional structure determined and the mechanisms by which this chaperone functions and is modulated are not yet fully known. Even the set of co-chaperones and post-translational modifications, that play key role in the processes aforementioned, are not yet complete understood. The obvious therapeutic potential that Hsp90 from human, and Hsp90 from other organisms, have, make essential to unravel the forces that stabilize their structure and the mechanisms by which their functions are modulated resulting in the maturation of proteins clients. The importance in studying Hsp90 brought together researchers involved in studying the relationship between structure and function of proteins, particularly chaperones from human, plants and protozoa, that will focus on the following goals: 1) Determination of the conformational aspects of Hsp90, its isolated domains and some of its co-chaperones. 2) Characterization of the ATPase activity of the various types of Hsp90 available. 3) Investigation of the interaction of Hsp90 with several co-chaperones, inhibitors and client proteins. 4) Investigation of the effect that post-translational modifications have in the structure and function of Hsp90. To show our commitment to this proposal we would like to stress that some studies are already underway and already have potential to generate knowledge that is both new and relevant. (AU)

Characterization and functional analysis of human SKD3 in yeast

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Scientific Initiation
Grant number:12/08294-0
Duration: July 01, 2012 - June 30, 2013
Abstract
Proteins are involved in many cellular processes and to protect the functional activity of these proteins, cells make use of a complex system which controls folding and protein degradation. This system is known as PQC (protein quality control) and comprises mainly the molecular chaperones and the proteasome system. Among several families of molecular chaperones, that of Hsp100 stands out for its activity in breaking down aggregated proteins and for its cooperation with the proteasome system. In bacteria, yeast and plants the knockout of the gene responsible for expression of Hsp100 causes increased thermosensitivity and lethality in stress conditions. Interestingly, although present in the organisms mentioned above and in organelles such as mitochondria, an equivalent of Hsp100 has not been found in animals. This project aims to test the overall ability of a human gene that is a Hsp100-like candidate to have a function similar to Hsp100, as complementation of a yeast strain deleted of the hsp104 gene. The identification of a protein in humans which function similar to Hsp100 has considerable interest because protein aggregates are involved in amyloidogenic diseases. (AU)

Using calorimetric techniques to characterize protein-protein interactions: applications in molecular chaperones

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Supervisor abroad: Conceição A. S. A. Minetti
Research place: Rutgers The State University of New Jersey, New Brunswick (United States)
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships abroad - Research Internship - Doctorate
Grant number:11/22212-4
Duration: April 01, 2012 - July 31, 2012
Abstract
Molecular chaperones, also described as heat shock proteins (Heat shock protein, Hsp) act preventing aggregation and incorrect folding of proteins. The incorrect protein folding causes loss of function, leading to diseases like Alzheimer's and Parkinson's disease and some cancers with subsequent decrease in life expectancy. The Hsp90 molecular chaperone is one of the most important, considered essential for cell viability in eukaryotes and is usually associated with proteins acting in the cycle and cell signaling. Moreover, recently it was implicated in the stabilization of the tumor phenotype of various cancers. The interaction with co-chaperones, considered auxiliary proteins of chaperones, enables that Hsp90 acts as a hub protein being a central point of regulation of several proteins and consequently of regulatory pathways. Many of the co-chaperones have one or more TPR domains that interact with the C-terminus of Hsp90. Recent results from our group show the mapping of amino acid residues involved in the interaction between the C-terminal domain of Hsp90 (Hsp90-C) and Tom70 TPR domain using the technique of cross-linking coupled with LC-MS/MS. Thus, the goal in this project is to use calorimetric techniques to characterize the thermodynamic interaction between these proteins based on our previous result. We expect with this, contribute to define the parameters of interaction and generate knowledge at the molecular level that allows the creation of strategies for intervention in the functioning of Hsp90, a target protein in the treatment of cancer. In addition, the client protein of Hsp90, p53, a tumor suppressor protein can, under specific conditions, aggregate and form amyloid fibrils. Aggregation experiments performed in our laboratory showed that Hsp90 was able to inhibit the aggregation of WT and p53 mutant R248Q. Therefore, this work also intend to investigate thermodynamic characteristics of the interaction between p53 (and mutant) and Hsp90. (AU)

Study of the interaction between Hsp90 and co-chaperones with TPR domain

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Master
Grant number:11/16047-0
Duration: April 01, 2012 - February 28, 2013
Abstract
Molecular chaperones, also known as heat shock proteins (Heat shock protein, Hsp) act to prevent aggregation and improper folding of proteins. The incorrect folding of the protein causes loss of function, as in some types of cancer and neurodegenerative diseases, and reduce life expectancy. These deleterious effects motivate the development of compounds that may, for instance, induce the expression or modulate the function of molecular chaperones. Thus, the study of the interaction between proteins and the search for compounds that can modulate the function of chaperones has become extremely important for the advancement of scientific knowledge on different cellular mechanisms and in searching for new drugs to increase the production of proteins properly folded. The chaperone Hsp90 is important for cell physiology, because it is involved in the stabilization of several proteins involved in signaling, and has recently been implicated in the stabilization of the tumor phenotype of various cancers. The interaction with co-chaperones, auxiliary proteins, enables Hsp90 to act as a hub, i.e. a central point of regulation of several proteins, and thus to regulate several pathways. The intervention in the interaction of Hsp90 co-chaperone is therefore a potentially effective strategy to intervene in the function of this chaperone. Many of these co-chaperones have a domain named TPR that interact with a specific MEEVD motif located in the C-terminus of Hsp90. Our goal is to use the technique of isothermal titration microcalorimetry (ITC) to characterize the thermodynamics of interactions between this peptide and several co-chaperones containing TPR domain to generate knowledge towards the development of intervention strategies in the interaction between these proteins. Our proposal is also supported on previous results from our group on the thermodynamic characterization of the interaction of the C-terminal domain of Hsp90 co-chaperone with a TPR. (AU)

Study of the molecular chaperone Hsp90 modulation through the characterization of interactions with co-chaperones, client protein, ligands and post-translational modifications

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Post-Doctorate
Grant number:11/18611-0
Duration: December 01, 2011 - January 31, 2013
Abstract
The biological function of proteins is related to its three dimensional structure acquired via protein folding process. In this context, the molecular chaperones play a key role acting as auxiliary protein on protein folding, refolding and dissociation of protein aggregates. Hsp90 is one of the most important molecular chaperones, is essential for cell viability in eukaryotes and is usually associated with proteins involved in cell cycling and cell signaling, which makes these chaperone a very interesting targeting for therapeutic approaches for several diseases. The chaperone activity of Hsp90 can be modulated by other proteins, called co-chaperones, client proteins, ligand interaction and post-translational modifications. Thus, the study of modulators of Hsp90 can help to elucidate major open questions about the function and regulation of Hsp90. The scope of this project is to investigate some co-chaperones and client proteins of Hsp90, involved in cell cycle and transcriptional regulation/chromatin remodeling, for example, the co-chaperone p23 and its variants, the co-chaperones Pih1 and Tah1 and protein clients as Wee1 and CDKs; and how these proteins can act as modulators of Hsp90 function, in addition to study of interaction with ligands such as curcumin a epigallocatechin gallate and characterization of post-translational modifications. The techniques applied for initial characterization are circular dichroism and fluorescence, for further characterization hydrodynamic methods, as dynamic light scattering, SEC-MALS and analytical ultracentrifugation will be utilized. Calorimetric methods as differential scanning calorimetry and isothermal titration calorimetry will be also used; and also the small-angle X-ray scattering technique. In this way, we intend to enhance our understanding of Hsp90 modulation. (AU)

Studies towards the potential application of Streptomyces in biocatalysis reactions

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Physical Sciences and Mathematics - Chemistry
Support type:Scholarships in Brazil - Scientific Initiation
Grant number:11/14910-3
Duration: October 01, 2011 - March 31, 2012
Abstract
Microorganisms, plants and animals produce a wide range of compounds that cold act as drugs, pigments, fragrances, flavorings and cosmetics. The biosynthesis of such metabolites is mediated by multienzymes complexes acting since the cabonic chain extension to the post-modifying steps. The versatility of such enzymes could be applicable to bioconversions of xenobiotics in order to obtain chiral blocks. We intend to study in this project the capacity of strptomyces strains to promote biotransformations mediated by monooxigenases, oxido-reductases and hydrolysis using whole cell systems. (AU)

Calorimetry researcher visiting

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Visiting researcher: Conceição A. Minetti
Visiting researcher institution: Rutgers The State University of New Jersey, New Brunswick (United States)
Field of knowledge:Biological Sciences - Biochemistry
Support type:Research Awards - Visiting Researcher Grant - International
Grant number:11/17643-6
Duration: September 22, 2011 - November 21, 2011
Abstract
The coordinator of this proposal, Carlos Ramos, has a research project financed by FAPESP, which has as its objective the use of the technique of isothermal titration calorimetry (ITC) to study the interaction between proteins and small molecules. The proteins studied in the research group are those involved in protein homeostasis, especially molecular chaperones and sirtuins. With the great experience prof Minetti has in the area of study of thermodynamics of interaction between proteins and ligands, she can make important contributions in our research project and training of specialized personnel. In addition to that, an automated ITC will be installed in an Institutional Laboratory at UNICAMP and will be available to researchers of universities in Sao Paulo state. Thus the training of specialized personnel will be very important to the widespread use of this equipment. (AU)

From toxic aggregates to properly folded proteins - functional characterization of a novel Hsp104 ortholog in mammals, and insight into the molecular architecture and substrate specificities of type i and Type II HSP40S

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Scholarships in Brazil - Post-Doctorate
Grant number:11/07320-5
Duration: September 01, 2011 - August 31, 2013
Abstract
The proper structure of proteins is maintained by a sophisticated system of proteinchaperones. When this system is disrupted, proteins can become unfolded, lose function,and form toxic aggregates that are implicated in a number of different pathologies. Theseaggregates can be eliminated by specialized chaperones that contain a disaggregaseactivity working in conjunction with an Hsp70 system that is itself regulated by differentHsp40 co-chaperones. To date, no disaggregase has been described in humans.Furthermore, precisely how different Hsp40s regulate Hsp70 activity is not fullyunderstood, although the orientation of a conserved J-domain within the structure isknown to be important. In this project, we propose to demonstrate for the first time adisaggregase activity for a human protein, SKD3, and determine what effects silencing itsexpression has on cells during high stress conditions. Furthermore, we will obtain highresolutionstructural information on wild-type and mutant type I and type II hsp40 cochaperonesfrom yeast by NMR TROSY, providing the first high resolution study on howthe central domain dictates the quaternary structure of the J domain. Furthermore, we alsowill identify novel substrates of human type I and type II Hsp40s, and provide insightinto the substrate specificities of each. The information obtained in these studies willsignificantly broaden our understanding of how chaperone systems cooperate to converttoxic protein aggregates into functional proteins, with specific emphasis on human andother mammalian chaperone systems. (AU)

Protein folding, stability and structure

Awardee:
Home Research Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Research Projects - Thematic Grants
Grant number:05/00462-8
Duration: April 01, 2006 - September 30, 2011
FAPESP publication about the research grant:http://www.fapesp.br/tematicos/saude_ramos.pdf
Abstract
Our yet little understanding of the protein folding process holds back the understanding of several cellular processes because the conversion of a backbone into a native protein is a key element in the translation of organism's genetic information. As the organism ages, the folding seems to deviates becoming a signal to several diseases (mainly neurodegenerative). Protein misfolding causes deposition in the cell in the form of aggregates or amyloidal fibrils, both of which has toxic effects. One way to cell protection is throughout molecular chaperones, which help protein folding and may help protein disaggregation. Therefore, chaperones seem to have a fundamental role in the organism by increasing the success of physiological functions and protecting cells of become ill. My proposal has the main objective of understand protein folding by: 1) studying the folding pathway and the stability of proteins, mainly globins; 2) characterizing the forces and the mechanisms of amyloidal fibril formation; 3) structural and functional characterization of chaperones and 4) studying the mechanisms by which chaperones help folding, stop aggregation, ressolubilize aggregates, and interact with proteins involved in cell malignization. These phenomena mutually take place in the cell and their study in conjunction as proposed here may increase our understanding of protein folding inside the cell, which will generate important new thinking that lead to new therapies. (AU)

The sugarcane EST project (SucEST) data mining proposal environmental stress: chaperones, HSPS and other stress related proteins

Awardee:
Home Research Institution: Laboratório Nacional de Luz Síncrotron (LNLS). Associação Brasileira de Tecnologia de Luz Síncrotron (ABTLuS). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas, SP, Brazil
Principal Investigator:Carlos Henrique Inacio Ramos
Field of knowledge:Biological Sciences - Biochemistry
Support type:Genome Research Awards
Grant number:00/07422-8
Duration: August 01, 2000 - July 31, 2002
Abstract
Some newly synthesized proteins require the assistance of molecular chaperones for their folding. Chaperones are also involved in the dissolution of protein aggregates. Hence, the study of molecular chaperones has biotechnology and medical importance. We intend to search the SUCEST data bank in order to identify and annotate the genes of stress related proteins, mainly chaperones and Hsps. (AU)
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