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Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design. publishersversion published

Made available in DSpace on 2022-04-28T19:47:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-28 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Leishmania developmental cycle comprises three main life forms in two hosts, indicating that the parasite is continually challenged due to drastic environmental changes. The disruption of this cycle is critical for discovering new therapies to eradicate leishmaniasis, a neglected disease that affects millions worldwide. Telomeres, the physical ends of chromosomes, maintain genome stability and cell proliferation and are potential antiparasitic drug targets. Therefore, understanding how telomere length is regulated during parasite development is vital. Here, we show that telomeres form clusters spread in the nucleoplasm of the three parasite life forms. We also observed that amastigotes telomeres are shorter than metacyclic and procyclic promastigotes and that in parasites with continuous in vitro passages, telomere length increases over time. These observed differences in telomere length among parasite’s life stages were not due to lack/inhibition of telomerase since enzyme activity was detected in all parasite life stages, although the catalysis was temperature-dependent. These data led us to test if, similar to other eukaryotes, parasite telomere length maintenance could be regulated by Hsp83, the ortholog of Hsp90 in trypanosomatids, and Leishmania (LHsp90). Parasites were then treated with the Hsp90 inhibitor 17AAG. The results showed that 17AAG disturbed parasite growth, induced accumulation into G2/M phases, and telomere shortening in a time-dependent manner. It has also inhibited procyclic promastigote’s telomerase activity. Besides, LHsp90 interacts with the telomerase TERT component as shown by immunoprecipitation, strongly suggesting a new role for LHsp90 as a parasite telomerase component involved in controlling telomere length maintenance and parasite life span. Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) Faculdade Brazileira Multivix Hemocentro da Faculdade de Medicina de Ribeirão Preto Universidade of São Paulo São Carlos Institute of Chemistry University of São Paulo Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP)

Made available in DSpace on 2020-12-12T01:46:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 In recent years, research has focused on the immunoreactive components of the Sporothrix schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In a previous study, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with an adjuvanted mixture of S. schenckii cell wall proteins. Here, we sought to assess the protective potential of a Sporothrix spp. recombinant enolase (rSsEno) formulated with or without the adjuvant Montanide Pet-GelA (PGA) against the S. brasiliensis infection in mice. Mice that were immunized with rSsEno plus PGA showed increased antibody titters against rSsEno and increased median survival time when challenged with S. brasiliensis as compared with mice that had not been immunized or that were immunized with rSsEno alone. Immunization with rSsEno plus PGA induced a predominantly T-helper 1 cytokine pattern after in vitro stimulation of splenic cells with rSsEno: elevated levels of IFN-γ and IL-2, as well as of other cytokines involved in host defense against sporotrichosis, such as TNF-alpha, IL-6, and IL-4. Furthermore, we show for the first time the presence of enolase in the cell wall of both S. schenckii and S. brasiliensis. As a whole, our results suggest that enolase could be used as a potential antigenic target for vaccinal purposes against sporotrichosis. São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Clinical Analysis São Carlos Institute of Chemistry University of São Paulo, P.O. Box 780 Núcleo Biotecnológico de Curauma (NBC) Pontificia Universidad Católica de Valparaíso Center of Biological and Health Sciences Federal University of São Carlos Oswaldo Cruz Foundation (Fiocruz) Evandro Chagas National Institute of Infectious Diseases (INI) Laboratory of Clinical Research on Dermatozoonoses in Domestic Animals (Lapclin-Dermzoo) São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Clinical Analysis

First described in yeast in 1932 by Christian & Warburg, the Old Yellow Enzyme (OYE) (EC 1.6.99.1) has aroused the interest of the scientific community regarding its high ability to catalyze stereoselective reactions of α/β-unsaturated carbonyl compounds with important industrial applications. In addition, the OYE family of proteins has been found in different organisms, such as plants, bacteria and protozoa, but not in mammals, which makes it an excellent candidate for a functional and molecular study aimed at more effective therapies with fewer undesirable side effects. Several OYE orthologues have been characterized; however, the real physiological role for most members of this family of proteins remains a mystery. In this paper, we present the structural studies of the OYE of Leishmania braziliensis. The findings are discussed in comparison with OYE of Trypanosoma cruzi, revealing some biophysical differences. The main differences are related to their chemical and thermal stabilities and behavior in solution. In addition, the L. braziliensis OYE shape is more elongated than that of the T. cruzi orthologue. Despite this, the active sites of these enzymes do not appear to have major differences, since their interactions with the substrate menadione occur with an affinity of the same order of magnitude, revealing that the binding sites in both proteins are essentially similar.

Made available in DSpace on 2021-06-25T11:52:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) CIHR Heat shock proteins (Hsps) are involved in several important aspects of the cell proteostasis. Hsp90 interacts with at least a tenth of the cell proteome helping a large number of proteins to fold correctly. Hsp90 function is modulated by several co-chaperones having TPR (tetratricopeptide repeat) domains that allow for interaction with the C-terminal MEEVD motif of the chaperone. Another important chaperone, Hsp70, has a C-terminal EEVD motif that binds to TPR. Leishmania is a protozoan that causes leishmaniasis, a neglected disease in humans and other animals. There is still no effective treatment for leishmaniasis, however the study of structure and function of the proteins of the parasite may generate potential targets for future therapeutic intervention studies. In this work, the genome of Leishmania major was searched for a novel TPR-domain gene, which is conserved in Leishmania. The recombinant protein, LmTPR, was produced in pure and folded state and was characterized by biophysical tools as a monomer with an elongated conformation. Studies in Leishmania major were also preformed to complement these in vitro experiments. Splice Leader RNA-seq analysis and Western blot indicated that the protein was expressed in all developmental stages of the parasite. Binding assays confirmed that both Hsp90 and Hsp70 bind specifically to LmTPR. Finally, sequence and structural predictions indicated a C terminal region as a RPAP3 domain. Altogether, this study identified a novel TPR-domain co-chaperone of Hsp90 that is conserved and expressed in all developmental stages of Leishmania major. (C) 2021 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Univ Campinas UNICAMP, Inst Chem, BR-13083970 Campinas, SP, Brazil Natl Inst Sci & Technol Struct Biol & Bioimage IN, Rio De Janeiro, RJ, Brazil Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil Univ Sao Paulo, Sao Carlos Inst Chem, Sao Carlos, SP, Brazil Univ Toronto, Dept Biochem, Toronto, ON M5G 1M1, Canada Univ Toronto, Dept Chem, Toronto, ON M5S 3H6, Canada Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil FAPESP: 2012/50161-8 FAPESP: 2017/26131-5 FAPESP: 2018/04375-2 FAPESP: 2014/25967-4 FAPESP: 2019/11496-3 CAPES: 99999.004913/2015-09 CIHR: PJT-173491

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The Cell Wall Integrity Pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence, and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA, and RlmA are Hsp90 clients and that a PkcA(G579R) mutation abolishes this interaction. PkcA(G579R) also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasizing the importance of this pathway in supporting thermotolerance and cell wall integrity.

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.

Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.

Molecular chaperones have several critical functions in protein metabolism. Among them, some are involved in processes that culminate in the extraction of entangled polypeptides from protein aggregates, releasing unfolded structures prone to be refolded or directed to degradation. This action avoids the effect of toxic aggregates on cells and tissues. Molecular chaperones belonging to the Hsp100 family are widely distributed from unicellular and sessile organisms up to fungi and plants, exerting key functions related to the reduction of the effects caused by different forms of stress. The Hsp100 proteins belong to the AAA+ (ATPases Associated with diverse cellular Activities) family and form multichaperone systems with Hsp70 and small Hsp chaperones families. However, Hsp100 are absent in metazoan, where protein disaggregation action is performed by a system involving the Hsp70 family, including Hsp110 and J-protein co-chaperones. Here, the structural and functional aspects of these protein disaggregation systems will be reviewed and discussed in the perspective of the Hsp100 system absent in the metazoan kingdom. This feature focuses on Hsp100 as a hot spot for drug discovery against human infectious diseases such as leishmaniasis and malaria, as Hsp100 is critical for microorganisms. The current data available for Hsp100 in Leishmania spp. and Plasmodium spp. are also reviewed.

Molecular chaperones and co-chaperones play an essential role in the life cycles of protozoa belonging to the genus Leishmania. The small glutamine-rich TPR-containing protein (SGT) is a co-chaperone that can be divided into three domains: N-terminal, tetratricopeptide (TPR) and C-terminal. The TPR domain is responsible for interactions with both Hsp70 and Hsp90; however, the mechanism of interaction and the functionality of SGT are unclear. In this context, we present the structural and functional characterization of Leishmania braziliensis SGT (LbSGT), aiming to elucidate how this co-chaperone interacts with the Hsp90/Hsp70 chaperone machinery. Structurally, the recombinant LbSGT behaves as an α-helical, multidomain and elongated dimer in solution. Despite their low amino acid sequence identity and similarity, LbSGT shares structural properties and domain organization with the Hsp70-interacting protein (HIP) co-chaperone. Functionally, LbSGT is a cognate protein in L. braziliensis promastigote cells and interacts indiscriminately, with similar affinities, with both Hsp90 and Hsp70 chaperones, capable of working as an adaptor protein. Sequence analysis indicates that LbSGT interacts via a dicarboxylate clamp, the same mechanism used by the Hsp90-Hsp70-organizing protein (HOP) co-chaperone. These results suggest that SGT can develop the same function as HOP but using the HIP structural scaffold.

In recent years, research has focused on the immunoreactive components of theS. schenckiicell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In previous studies, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with purified fungal wall proteins and adjuvants. In this study, the immunolocalization of this immunogen in the cell wall ofS. schenckiiandS. brasiliensisis shown for the first time. In addition, a recombinant enolase ofSporothrixspp (rSsEno) was studied with the adjuvant Montanide Pet-GelA (PGA) as a vaccine candidate. The rSsEno was produced with high purity. In addition, mice immunized with rSsEno plus PGA showed increased antibody titers against enolase and increased median survival time comparedto nonimmunized or rSsEno-immunized mice. Enolase immunization induced a predominant T-helper-1 (Th1) cytokine pattern in splenic cells after in vitro stimulation with rSsEno. Elevated production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) was observed with other cytokines involved in the innate immune defense, such as TNF-alpha, IL-6, and IL-4, which are necessary for antibody production. These results suggest that we should continue testing this antigen as a potential vaccine candidate against sporotrichosis.

Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

HOP is a cochaperone belonging to the foldosome, a system formed by the cytoplasmic Hsp70 and Hsp90 chaperones. HOP acts as an adapter protein capable of transferring client proteins from the first to the second molecular chaperone. HOP is a modular protein that regulates the ATPase activity of Hsp70 and Hsp90 to perform its function. To obtain more detailed information on the structure and function of this protein, we produced the recombinant HOP of Plasmodium falciparum (PfHOP). The protein was obtained in a folded form, with a high content of α-helix secondary structure. Unfolding experiments showed that PfHOP unfolds through two transitions, suggesting the presence of at least two domains with different stabilities. In addition, PfHOP primarily behaved as an elongated dimer in equilibrium with the monomer. Small-angle X-ray scattering data corroborated this interpretation and led to the reconstruction of a PfHOP ab initio model as a dimer. Finally, the PfHOP protein was able to inhibit and to stimulate the ATPase activity of the recombinant Hsp90 and Hsp70-1, respectively, of P. falciparum. Our results deepened the knowledge of the structure and function of PfHOP and further clarified its participation in the P. falciparum foldosome.

Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence-labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell-based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP-binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.

Hep1 is a mitochondrial Hsp70 (mtHsp70) co-chaperone that presents a zinc finger domain essential for its function. This co-chaperone acts to maintain mtHsp70 in its soluble and functional state. In this work, we have demonstrated that Leishmania braziliensis mtHsp70 (LbmtHsp70) is also dependent on the assistance of Hep1. To understand the L. braziliensis Hep1 (LbHep1) structure-function relationship, we produced LbHep1 and two truncated mutants corresponding to the C-terminal zinc finger domain and the N-terminal region. We observed that LbHep1 is composed of an unfolded N-terminal region and a β-sheet-folded C-terminal domain, which holds the zinc-binding motif. Both LbHep1 and the zinc finger domain construction maintained LbmtHsp70 solubility in co-expression systems after cell lysis. In solution, LbHep1 behaved as a highly elongated monomer, probably due to the unfolded N-terminal region. Furthermore, we also observed that the zinc ion interacted with LbHep1 with high affinity and was critical for LbHep1 structure and stability because its removal from LbHep1 solutions altered the protein structure and stability. In vitro, LbHep1 protected, in sub-stoichiometric fashion, LbmtHsp70 from thermally induced aggregation but did not present intrinsic chaperone activity on model client proteins. Therefore, LbHep1 is a specific chaperone for LbmtHsp70.

The p23 proteins are small acidic proteins that aid the functional cycle of the Hsp90 molecular chaperone. Such co-chaperone acts by temporarily inhibiting the ATPase activity of Hsp90 and exhibits intrinsic chaperone activity, suggesting independent roles. A search for p23 in the Plasmodium falciparum genome led to the identification of two putative proteins showing 13% identity to each other and approximately 20% identity to human p23. To understand the presence of two p23 proteins in this organism, we generated recombinant p23 proteins (Pfp23A and Pfp23B) and investigated their structure and function. The proteins presented some similarities and dissimilarities in structural contents and showed different chemical and thermal stabilities, with Pfp23A being more stable than Pfp23B, suggesting that these proteins may present different functions in this organism. Both Pfp23 proteins behaved as elongated monomers in solution and were capable of preventing the thermal-induced aggregation of model client proteins with different efficiencies. Finally, the Pfp23 proteins inhibited the ATPase activity of recombinant P. falciparum Hsp90 (PfHsp90). These results validate the studied proteins as p23 proteins and co-chaperones of PfHsp90.

Heat shock protein 70 kDa (Hsp70) is a conserved molecular chaperone family involved in several functions related to protein homeostasis. In eukaryotes, Hsp70 homologues are found in all cell compartments. The mitochondrial Hsp70 isoform (mtHsp70) is involved in import of mitochondrial matrix proteins as well as their folding and maturation. Moreover, mtHsp70 has the propensity to self-aggregate, and it depends on the action of the co-chaperone Hsp70-escort protein 1 (Hep1) to be produced functional. Here, we analyze the solution structure and function of mtHsp70 of Leishmania braziliensis (LbmtHsp70). This recombinant protein was obtained folded, in the monomeric state and it has an elongated shape. We observed that LbmtHsp70 suffers thermal aggregation that depends on the protein concentration and is composed of domains with different thermal stabilities. LbmtHsp70 interacted with adenosine nucleotides with a thermodynamic signature different from those reported for human orthologues and interacted, driven by both enthalpy and entropy, with L. braziliensis Hep1 (LbHep1) with a nanomolar dissociation constant. Moreover, LbHep1 stimulated the LbmtHsp70 ATPase activity. Since little is known about mitochondrial Hsp70, particularly in protozoa, we believe that our data are of interest for understanding protozoan Hsp70 machinery.

Heat shock protein of 90kDa (Hsp90) is an essential molecular chaperone involved in a plethora of cellular activities which modulate protein homeostasis. During the Hsp90 mechanochemical cycle, it undergoes large conformational changes, oscillating between open and closed states. Although structural and conformational equilibria of prokaryotic and some eukaryotic Hsp90s are known, some protozoa Hsp90 structures and dynamics are poorly understood. In this study, we report the solution structure and conformational dynamics of Leishmania braziliensis Hsp90 (LbHsp90) investigated by small angle X-ray scattering (SAXS). The results indicate that LbHsp90 coexists in open and closed conformations in solution and that the linkers between domains are not randomly distributed. These findings noted interesting features of the LbHsp90 system, opening doors for further conformational studies of other protozoa chaperones.

The Clp/Hsp100 AAA+ chaperone family is involved in recovering aggregated proteins and little is known about other orthologs of the well studied ClpB from Escherichia coli and Hsp104 from Saccharomyces cerevisiae. Plant Hsp101 is a good model for understanding the relationship between the structure and function of Hsp100 proteins and to investigate the role of these chaperones in disaggregation processes. Here, we present the cloning and purification of a sugarcane ortholog, SHsp101, which is expressed in sugarcane cells and is a folded hexamer that is capable of binding nucleotides. Thus SHsp101 has the structural and functional characteristics of the Clp/Hsp100 AAA+ family.

The interest in analytical ultracentrifugation (AUC) to analyze protein structural parameters and interactions has increased in the past decades as a result of several developments on new generation instrumentation and data analysis tools. In this article, we review AUC principles and applications to study proteins, emphasizing molecular targets of Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the most common infectious diseases known to man and responsible for millions of human deaths in the world. The increasing incidence of TB in developing countries, the proliferation of multidrug resistant strains, and the absence of resources for treatment have highlighted the need of developing new drugs against TB. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of aromatic amino acids. This pathway is absent from mammals and shown to be essential for the survival of Mycobacterium tuberculosis, the causative agent of TB. Accordingly, enzymes of aromatic amino acid biosynthesis pathway represent promising targets for structure-based drug design. The first reaction in phenylalanine biosynthesis involves the conversion of chorismate to prephenate, catalyzed by chorismate mutase. The second reaction is catalyzed by prephenate dehydratase (PDT) and involves decarboxylation and dehydratation of prephenate to form phenylpyruvate, the precursor of phenylalanine. Here, we describe utilization of different techniques to infer the structure of M. tuberculosis PDT (MtbPDT) in solution. Small angle X-ray scattering and ultracentrifugation analysis showed that the protein oligomeric state is a tetramer and MtbPDT is a flat disk protein. Bioinformatics tools were used to infer the structure of MtbPDT. A molecular model for MtbPDT is presented and molecular dynamics simulations indicate that MtbPDT is stable. Experimental and molecular modeling results were in agreement and provide evidence for a tetrameric state of MtbPDT in solution.

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90.

Tropical diseases affect the lives of at least one-tenth of the global population and are among the main global health priorities of the World Health Organization, the United Nations branch concerned with international public health. Most tropical diseases are caused by blood and tissue protozoal parasites, such as those belonging to the Plasmodium and Leishmania genera. Specifically, Leishmania spp. cause human leishmaniasis, and Plasmodium spp. cause malaria. Due to their overlapping geographical regions, these parasites are of great public health concern. They are microscopic, unicellular eukaryotes that are transmitted by blood-sucking insects, and this transmission can be stressful for both the parasites and the host. To cope with this stress, the parasites cycle between different stages in which many proteins are involved. Most of these proteins require assistance to reach their correct conformations; therefore, they are aided by molecular chaperones and Heat Shock Proteins (Hsps). Hsp70 and Hsp90 are the most important Hsps that assist in folding, and they are part of a Protein Quality Control system that helps maintain protein homeostasis. Furthermore, their functions in protozoa have expanded, as both Hsp70 and Hsp90 chaperones play important roles in cell growth and adaptation, allowing life cycle progression. Interaction networks of Hsp70 and Hsp90 in the Plasmodium and Leishmania genera are presented in this chapter.

The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins' interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90.

The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing “client” proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (kcat = 0.320 min− 1) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC50 of 0.7 μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis.

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50°C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of ∼63–64°C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents. Proteins 2008. © 2008 Wiley-Liss, Inc.

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study.

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 angstrom resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event. (c) 2005 Elsevier Inc. All rights reserved.

Background Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins of the yeast cytosol. Sis1 is an essential gene whereas the ydj1 gene is essential for growth at elevated temperatures and cannot complement sis1 gene deletion. Truncated polypeptides capable of complementing the sis1 gene deletion comprise the J-domain of either Sis1 or Ydj1 connected to the G/F region of Sis1 (but not Ydj1). Sis1 mutants in which the G/F was deleted but G/M maintained were capable of complementing the sis1 gene deletion. Results To investigate the relevance of central domains on the structure and function of Ydj1 and Sis1 we prepared Sis1 constructs deleting specific domains. The mutants had decreased affinity for heated luciferase but were equally capable of stimulating ATPase activity of Hsp70. Detailed low resolution structures were obtained and the overall flexibility of Hsp40 and its mutants were assessed using SAXS methods. Deletion of either the G/M or the G/M plus CTDI domains had little impact on the quaternary structure of Sis1 analyzed by the SAXS technique. However, deletion of the ZFLR-CTDI changed the relative position of the J-domains in Ydj1 in such a way that they ended up resembling that of Sis1. The results revealed that the G/F and G/M regions are not the only flexible domains. All model structures exhibit a common clamp-like conformation. Conclusions Our results suggest that the central domains, previously appointed as important features for substrate binding, are also relevant keeping the J-domains in their specific relative positions. The clamp-like architecture observed seems also to be favorable to the interactions of Hsp40 with Hsp70.

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether, LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action.

O estudo do processo de tradução no metabolismo celular atrai o interesse de vários grupos, em particular, o estudo do 21o aminoácido, a selenocisteína. A incorporação da selecisteína foi descrita em Escherichia coli e recentemente em eucariotos. O primeiro passo desta via é iniciado pela Seril-tRNA Sintetase que aminoacila o Ser-tRNASec (SelC) com uma serina. Em E. coli, o segundo passo é realizado pela Sec-sintetase (SelA) que remove o grupo hidroxil da cadeia lateral da serina, formando um intermediário aminoacrilil. Este serve como aceptor de seleno-fosfato gerando a selenocisteína. Em eucariotos, o processo análogo é realizado pela PSTK e pela SepSecS, que fosforila e seleniza a serina respectivamente. Interessados nesta parte da via, iniciamos estudos moleculares das enzimas Fosfoseril-tRNA Kinase de Trypanosoma brucei e Leishmania major e Seril-tRNA Sintetase de Trypanosoma brucei. Para o gene da enzima Fosfoseril-tRNA Kinase de T. brucei não foi possível obter um clone sem mutação. Já o gene da enzima Fosfoseril-tRNA Kinase de L. major foi clonado em vetor pET28 e a enzima foi expressa em células de E. coli porém com baixo rendimento impedindo a continuidade dos experimentos planejados. Portanto passou-se a investigar a enzima envolvida no primeiro passo da via, no caso, a Seril-tRNA Sintetase de T. brucei. Esta já se encontrava clonada e expressando em E. coli na fração solúvel. A proteína recombinante foi purificada com precipitação com 60% de sulfato de amônio e resinas de hidrofobicidade e de afinidade por níquel. Experimentos de gel nativo, DLS e fluorescência de anisotropia revelaram que, após a purificação, a enzima permanece estável e livre de agregações, possuindo um raio hidrodinâmico de 4,32nm e massa molecular de 110kDa. Acima de 150nM de proteína, ela encontra-se inteiramente na forma dimérica. Estabelecidos estes parâmetros, informações sobre a ligação com o Ser-tRNASec poderão ser obtidos a partir da técnica de anisotropia de fluorescência visto que experimentos iniciais realizados com a SerRS adicionando-se o Ser-tRNASec mostraram-se promissores. The translation process study is central role in the cellular metabolism and attracts the interest of several groups, in particular, the study of the 21º amino acid, the selenocystein. The selenocystein incorporation pathway was described in Escherichia coli and recently in eukaryotes. The first step of this pathway is initiated by Seryl-tRNA Synthetase that aminoacilates the Ser-tRNASec (SELC) with serine. In E. coli, the second step is performed by the Sec-synthase (SELA) that removes hydroxyl group of the serine side chain, forming an aminoacrylil intermediary. This serves as an acceptor of seleno phosphate generating the selenocystein. In eukaryotes, the similar process is performed by PSTK and SepSecS, which phosphorylate serine and adds the selenium, respectively. Interested in this pathway, we performed initial molecular studies of the Phosphoseryl-tRNA synthetase of Trypanosoma brucei and Leishmania major and Seryl-tRNA synthetase of Trypanosoma brucei. The gene that encodes T. brucei Phosphoseryl-tRNA synthetase was obtained with several mutations. However, the gene encoding the T. brucei Phosphoseryl-tRNA synthetase was cloned into pET28 vector and the enzyme was expressed in E. coli cells, however at low amounts hampering the intended experiments. Therefore we initiated the investigation of the enzyme involved in the first step of this pathway, the Seryl-tRNA Synthetase from T. brucei. The enzyme was already cloned and expressing in the soluble fraction of E. coli. The recombinant protein was purified using 60% ammonium sulfate precipitation, hydrophobic and nickel affinity chromatography. Native gel experiments, DLS and anisotropy fluorescence was performed and allowed to conclude that, after purification, the enzyme remains stable and free of aggregation, with a hydrodynamic radius of 4.32 nm, molecular weight of 110kDa. Above 150nM protein its entirely in the dimeric form. Information about Ser-tRNASec binding can now be obtained from the technique of anisotropy seen that initial experiments with SerRS add Ser-tRNASec be shown to be promising.