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Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

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

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role. Electronic supplementary material The online version of this article (10.1007/s12192-020-01134-9) contains supplementary material, which is available to authorized users.

Sporotrichosis is a cosmopolitan mycosis caused by pathogenic species of Sporothrix genus, that in Brazil is often acquired by zoonotic transmission involved infected cats with S. brasiliensis. Previous studies showed that the Sporothrix spp. recombinant enolase (rSsEno), a multifunctional protein with immunogenic properties, could be a promising target for vaccination against sporotrichosis in cats. Nevertheless, the considerable sequence identity (62%) of SsEno with its feline counterpart is a great concern. Here, we report the identification in silico, chemical synthesis and biological validation of six peptides of SsEno with low sequence identity to its cat orthologue. All synthesized peptides exhibit B-cell epitopes on the molecular surface of SsEno and proved to be highly reactive with the serum of infected mice with S. brasiliensis and sera of cats with sporotrichosis. Interestingly, our study revealed that anti-peptide sera did not react with the recombinant enolase from Felis catus (cats, rFcEno), thus, may not trigger autoimmune response in these felines if used as a vaccine antigen. The immunization with peptide mixture (PeptMix) formulated with Freund adjuvant (FA), induced high levels of antigen-specific IgG, IgG1 and IgG2b antibodies that conferred protection upon passive transference in infected BALB/c mice with S. brasiliensis. We also observed, that the FA+PeptMix formulation induced a Th1/Th2/Th17 cytokine profile ex vivo, associated with protecting effect against the experimental sporotrichosis. Our results suggest that the six SsEno-derived peptides here evaluated, could be used as safe antigens for the development of vaccine strategies against feline sporotrichosis, whether prophylactic or therapeutic.

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

Made available in DSpace on 2019-10-04T12:37:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp9Os are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp9Os from different organisms have peculiarities despite their high sequence identity. Therefore, a detailed comparative analysis of several Hsp90 proteins is relevant to the overall understanding of their activity. Accordingly, the goal of this work was to evaluate the interaction of either ADP or ATP with recombinant Hsp9Os from different organisms ( human et and (3 isoforms, Plasmodium falciparum, Leishmania braziliensis, yeast and sugarcane) by isothermal titration calorimetry. The measured thermodynamic signatures of those interactions indicated that despite the high identity among all Hsp90s, they have specific thermodynamic characteristics. Specifically, the interactions with ADP are driven by enthalpy but are opposed by entropy, whereas the interaction with ATP is driven by both enthalpy and entropy. Complimentary structural and molecular dynamics studies suggested that specific interactions with ADP that differ from those with ATP may contribute to the observed enthalpies and entropies. Altogether, the data suggest that selective inhibition may be more easily achieved using analogues of the Hsp90-ADP bound state than those of Hsp90-ATP bound state. (C) 2019 Elsevier B.V. All rights reserved. Univ Sao Paulo, Sao Carlos Inst Chem, BR-13566590 Sao Carlos, SP, Brazil Univ Fed Sao Carlos, Ctr Biol & Hlth Sci, BR-13560970 Sao Carlos, SP, Brazil Univ Sao Paulo, Sao Carlos Inst Phys, BR-13560970 Sao Carlos, SP, Brazil Sao Paulo State Univ, Multiuser Ctr Biol Innovat CMIB, Biosci Languages & Exact Sci Inst, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Univ Campinas UNICAMP, Inst Chem, BR-13083970 Campinas, SP, Brazil Sao Paulo State Univ, Multiuser Ctr Biol Innovat CMIB, Biosci Languages & Exact Sci Inst, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2009/53989-4 FAPESP: 2011/23110-0 FAPESP: 2012/50161-8 FAPESP: 2013/25646-0 FAPESP: 2014/07206-6 FAPESP: 2015/26722-8 FAPESP: 2017/07335-9 FAPESP: 2017/18173-0 CNPq: 471415/2013-8 CNPq: 303129/2015-8

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 2021-06-25T10:49:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Prostate cancer is one of the most frequent neoplasms, associated with high mortality. Some factors may intensify its aggressiveness, for example, the overexpression of the epidermal growth factor receptor (EGFR) in some subtypes of prostate tumors. In this context, the inhibition of EGFR helps to fight neoplasia, with the use of the chimeric anti-EGFR monoclonal antibody, cetuximab (CTX). For cancer treatment in the metastatic stage, chemotherapy using docetaxel (DTX) is widely employed, however the drug is very liposoluble, hampering its bioavailability. In this sense, liquid crystalline systems have great potential for the delivery of DTX. Particularly, liquid crystalline nanodispersions can be used in parenteral routes, with many advantages, including the possibility of prolonged drug release, improved pharmacokinetics, allowing passive tumor accumulation by the EPR effect. Thus, the objective of this work was to develop a liquid crystalline nanodispersion (LCN) based on ethoxylated cetyl alcohol 20 and propoxylated 5 as surfactant (Procetyl®), oleic acid, DSPE-PEG-MAL (anchor for CTX binding) and soy phosphatidylcholine as oily phase and 1.5 % poloxamer dispersion in PBS buffer as an aqueous phase. The formulation was loaded with DTX and covalently functionalized with CTX to evaluate its cytotoxic potential against prostate cancer cells. The results suggested that the obtained system has a predominantly hexagonal crystalline phase, was able to be nanodispersed with low polydispersity, and presented negative zeta potential. CTX did not have its structure compromised after thiolation and was successfully covalently linked to LCN. In a prostate cancer cells, PC3, LCNs functionalized with CTX underwent greater cell uptake, resulting in greater cytotoxicity, compared to free DTX and non-functionalized DTX-loaded LCNs. Therefore, the present study reports for the first time an EGFR-targeted liquid crystal nanodispersion for selective to deliver DTX to prostate cells School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines Federal University of Ceará College of Pharmacy Dentistry and Nursing Department of Pharmacy University of International Integration of the Afro-Brazilian Lusophony Institute of Health Sciences São Carlos Institute of Chemistry University of São Paulo, São Carlos School of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo Ribeirão Preto School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines CNPq: # 465687/2014-8 FAPESP: #2014/50928-2

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

Made available in DSpace on 2022-04-29T08:32:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The structural fragility of monoclonal antibodies (mAbs), such as bevacizumab (BVZ), is a critical parameter for oral administration and can limit the use of several technologies to produce oral nanocarriers for these biomolecules. Nanoparticles (NPs) based on gellan gum (GG) and retrograded starch (RS) were rationally designed through polyelectrolyte complexation, and ionic cross-linking was exploited as an additional technological strategy to modulate the properties of nanocarriers. According to static light scattering analysis, the molecular weights of GG and RS were approximately 158 kDa and 1803 kDa, respectively. The influence of pH on the zeta potential (ZP) of polymers allowed the selection of pH 6.2 as the most suitable pH for the complexation of these polyelectrolytes. Non-cross-linked NPs were prepared at different polymer:drug ratios and the effects of formulation variables (polyelectrolyte ratio, drug and cross-linker concentrations, and polymer:drug ratio) on the formation and properties (size, ZP, and PDI) of cross-linked NPs were evaluated using a 33 full-factorial design. The average size of non-cross-linked and cross-linked NPs ranged from 260.1 - 299.6 nm to 265.7–629.9 nm, respectively. NPs-negative ZP (>- 20 mV) and high association efficiency (AE%) (>56.16%) were achieved. Cross-linking significantly increased the BVZ AE% (85%–100%). Analyses by attenuated total reflectance-Fourier transform infrared, fluorescence, circular dichroism, and differential scanning microcalorimetry techniques demonstrated that polyelectrolyte complexation and ionic cross-linking did not denature the secondary and tertiary structures of BVZ. The results revealed the suitability of the technological approaches used to produce BVZ-loaded nanocarriers with tailored properties, representing a potential platform for the oral delivery of BVZ. UNESP - São Paulo State University School of Pharmaceutical Sciences Department of Drugs and Medicines UNESP - São Paulo State University Institute of Chemistry Department of Analytical Chemistry Physical Chemistry and Inorganic USP - São Paulo University São Carlos Institute of Chemistry Department of Chemistry and Molecular Physics UNESP - São Paulo State University School of Pharmaceutical Sciences Department of Drugs and Medicines UNESP - São Paulo State University Institute of Chemistry Department of Analytical Chemistry Physical Chemistry and Inorganic CAPES: 001 FAPESP: 2014/50928-2 FAPESP: 2015/21412-0 FAPESP: 2017/16324-0 CNPq: 465687/2014-8

Made available in DSpace on 2021-06-25T11:06:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-20 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Prostate cancer (PC) is the second type of cancer, causing the greatest number of deaths among the male population in the world. An attractive approach to treat PC involves the superficial modification of liposomes with specific ligands to overexpressed receptors on the surfaces of tumor cells, such as transferrin receptors (TFR's). To our knowledge, this is the first time that transferrin-functionalized liposomes for delivery of DTX to prostate cancer cells were investigated. In this study, we developed and characterized docetaxel- loaded liposomes functionalized with transferrin (LIP-DTX-TF) and evaluated their effect on PC cell lines. The results showed that the systems had a nanometric size and the presence of DTX contributed to the reduction of the vesicle size and liposomes were stable. The encapsulation efficiency of DTX was approximately 69 and 37 % for liposomes with and without functionalization, respectively. The functionalization efficiency using TF was 31 %. The functionalization process did not affect the integrity of TF. DTX encapsulated in the liposomes showed slow and sustained release for LIP-DTX and LIP-DTX-TF (51.70 % and 31.97 %, respectively). The in vitro cytotoxicity assay in PC-3 and PNT2 cell lines suggest that LIP-DTX-TF was more toxic to PC-3 than the commercial formulation. Confocal microscopy proved that functionalized liposomes were successfully internalized in PC-3 cells. Furthermore, the interaction of these systems with the TFR's overexpressed on the cell surface was further corroborated using Quartz Crystal Microbalance with Dissipation technology (QCM-D). Therefore, although additional studies in three-dimensional cell models and in vivo xenograft studies are warranted, it can be concluded that the systems developed here are promising candidates for the delivery of docetaxel to prostate cancer cells. School of Pharmaceutical Sciences São Paulo State University (UNESP) College of Pharmacy Dentistry and Nursing Federal University of Ceara (UFC) School of Pharmaceutical Science of Ribeirão Preto University of São Paulo (USP) São Carlos Institute of Chemistry University of São Paulo (USP) School of Pharmacy and Pharmaceutical Sciences Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Biochemistry and Immunology Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Medicine Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Pharmaceutical Sciences São Paulo State University (UNESP) CNPq: # 465687/2014-8 FAPESP: #2014/50928-2

Human 71 kDa heat shock cognate protein (HSPA8, also known as Hsc70, Hsp70-8, Hsc71, Hsp71 or Hsp73) is a constitutively expressed chaperone that is critical for cell proteostasis. In the cytosol, HSPA8 plays a pivotal role in folding and refolding, facilitates protein trafficking across membranes and targets proteins for degradation, among other functions. Here, we report an in solution study of recombinant HSPA8 (rHSPA8) using a variety of biophysical and biochemical approaches. rHSPA8 shares several structural and functional similarities with others human Hsp70s. It has two domains with different stabilities and interacts with adenosine nucleotides with dissociation constants in the low micromolar range, which were higher in the presence of Mg2+. rHSPA8 showed lower ATPase activity than its homolog HSPA5/hGrp78/hBiP, but it was 4-fold greater than that of recombinant HSPA1A/hHsp70-1A, with which it is 86% identical. Small angle X-ray scattering indicated that rHSPA8 behaved as an elongated monomeric protein in solution with dimensions similar to those observed for HSPA1A. In addition, rHSPA8 showed structural flexibility between its compacted and extended conformations. The data also indicated that HSPA8 has capacity in preventing the aggregation of model client proteins. The present study expands the understanding of the structure and activity of this chaperone and aligns with the idea that human homologous Hsp70s have divergent functions.

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.

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.

Esta pesquisa examinou a influência de Instituições de Ensino Superior (IES) em grupos de startups no Brasil. Primeiramente, a literatura foi organizada em trabalhos que empregaram metodologias tradicionais e outros que utilizaram Análise de Redes Sociais (ARS). Em seguida, foram selecionadas fontes de dados disponíveis na web, formas de obtê-las e armazená-las. Após isso, foi realizado um exame a nível nacional dos tipos de atividades empreendedoras e a localização geográfica dos polos de startups. Finalmente, empregou-se ARS em uma rede alumni formada por fundadores de startups para medir o impacto das IES nessa estrutura. O exame das atividades empreendedoras revelou que as variações regionais poderiam estar favorecendo determinados setores e gerando oportunidades. Embora as startups se concentrem em polos economicamente mais desenvolvidas como as capitais, existem polos fora delas e também startups distribuídas em várias localidades. Isso fornece evidências de que o empreendedorismo se espalha facilmente pelo país. Quando se considera uma estrutura de conexões sociais cognitivas, como é o caso da rede alumni, a distância geográfica não impede que os atores se relacionem pois as ligações dependem mais da similaridade de assuntos e de trocas de conhecimentos do que da proximidade geográfica. Por isso, o alcance de influência das IES chega a limites inter-municipais entre 250 e 500 km e até inter-regionais em alguns casos, com distâncias acima de 1000 km. Observou-se, através da investigação da estrutura da rede, que a função educacional é distribuída entre universidades, faculdades e centros universitários, considerando IES públicas e privadas, e cada elemento apresenta diferentes formas de interagir com a rede de startups. Embora o Índice Geral de Cursos (IGC) seja um importante instrumento de avaliação da qualidade das IES no Brasil ele apresentou uma correlação fraca com a centralidade de grau da IES na rede. Isso significa que existem outros fatores que influenciam o posicionamento das IES dentro da estrutura e a formação do cluster ao seu redor. A escolaridade dos fundadores também é um elemento que merece atenção. Por exemplo, o período de graduação representa um importante momento na formação do empreendedor porque corresponde a maior parte das titulações. Os fundadores também continuam procurando aprofundar-se nos estudos mesmo após a criação da startup, por isso, o sistema de ensino superior deve estar preparado para recebê-los e atender da melhor forma possível suas expectavas e necessidades. Esta pesquisa busca justificar a importância da rede de ensino superior dentro da infraestrutura de suporte ao empreendedorismo brasileiro e pode influenciar políticas públicas como, por exemplo, o programa “Future-se” do Ministério da Educação que traz novas perspectivas sobre a função do ensino e pesquisa no Brasil, principalmente das universidades federais. De forma geral, este trabalho contribui para educação, empreendedorismo e políticas públicas. This research examined the influence of Higher Education Institutions (HEI) in groups of startups in Brazil. First, the literature was organized in works that used traditional methodologies and others that used Social Network Analysis (SNA). Then, data sources available on the Web, ways to obtain and store them were selected. After that, an examination was carried out at the national level of the types of entrepreneurial activities and the geographic location of the startups’ hubs. Finally, SNA was used in an alumni network formed by the founders of startups to measure the impact of HEIs on this structure. The examination of entrepreneurial activities revealed that regional variations could be favoring certain sectors and generating opportunities. Although startups are concentrated in more economically developed hubs such as capital cities, there are hubs outside of them and startups distributed in several locations. This provides evidence that entrepreneurship spreads easily across the country. When considering a structure of cognitive social connections, as in the alumni network’s case, the geographical distance does not prevent the actors from relating because the connections depend more on the similarity of subjects and the exchange of knowledge than on geographic proximity. Thus, the range of influence of HEIs reaches inter-municipal limits between 250 and 500 km and even inter-regional in some cases, with distances above 1000 km. Through the investigation of the network structure, it was observed that the educational function is distributed among universities, colleges, and university centers, considering public and private HEIs, and each element presents different ways of interacting with the startups’ network. Although the General Index of Courses (GIC) is an important instrument for assessing the quality of HEIs in Brazil, it showed a weak correlation with the centrality of HEI degree in the network. This means that other factors influence the position of the HEI within the structure and the formation of the cluster around it. The education of the founders is also an element that deserves attention. For example, the period at college represents an important moment in the entrepreneur’s formation because it represents the largest share of the degrees in the analysis. The founders also continue to seek to deepen their studies even after creating a startup; therefore, the higher education system must be prepared to receive them and meet their expectations and needs in the best possible way. This research seeks to justify the importance of the higher education network within the infrastructure supporting Brazilian entrepreneurship. It can influence public policies, such as the Ministry of Education’s “Future-se” program that brings new perspectives on the role of teaching and research in Brazil, mainly for federal universities. In general, this work contributes to education, entrepreneurship, and public policies.

Made available in DSpace on 2020-12-12T02:11:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Prostate cancer is the second cause of cancer death in men worldwide. Docetaxel (DTX), an antimitotic drug, is widely used for the treatment of metastatic prostate cancer patients. Taxotere® is a commercial DTX formulation. It contains a polysorbate 80 surfactant to improve DTX aqueous solubility, which has been associated with hypersensitivity reactions in patients. Liposomes have been used as promising delivery systems for a range of hydrophobic drugs, such as DTX, offering improved drug water solubility and biocompatibility, without compromising its anticancer activity. Herein, DTX-loaded liposomes were developed using the Box-Behnken factorial design. The optimized formulation was nano-sized, homogenous in size (67.47 nm) with high DTX encapsulation efficiency (99.95 %). The encapsulated DTX was in a soluble amorphous state, which was slowly released. Next, to increase the liposomes selectivity to prostate cancer cells, cetuximab, an anti-EGFR monoclonal antibody. was successfully conjugated to the surface of liposomes, without compromising cetuximab protein structure and stability. As expected, our results showed higher cellular uptake and toxicity of immunoliposomes, compared to non-targeted liposomes, in DU145 (EGFR-overxpressing) prostate cancer cells. To the best of our knowledge, this is the first report of engineering EGFR-targeted liposomes to enhance the selectivity of DTX delivery to EGFR-positive prostate cancer cells. Federal University of Ceará College of Pharmacy Dentistry and Nursing Department of Pharmacy School of Pharmacy Queen's University Belfast, 97 Lisburn Rd School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines University for International Integration of the Afro-Brazilian Lusophony Institute of Health Sciences College of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo São Carlos Institute of Chemistry University of São Paulo School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines

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.

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.

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.

The Hsp70 family of heat shock proteins plays a critical function in maintaining cellular homeostasis within various subcellular compartments. The human mitochondrial Hsp70 (HSPA9) has been associated with cellular death, senescence, cancer and neurodegenerative diseases, which is the rational for the name mortalin. It is well documented that mortalin, such as other Hsp70s, is prone to self-aggregation, which is related to mitochondria biogenesis failure. Here, we investigated the assembly, structure and function of thermic aggregates/oligomers of recombinant human mortalin and Hsp70-1A (HSPA1A). Summarily, both Hsp70 thermic aggregates have characteristics of supramolecular assemblies. They display characteristic organized structures and partial ATPase activity, despite their nanometric size. Indeed, we observed that the interaction of these aggregates/oligomers with liposomes is similar to monomeric Hsp70s and, finally, they were non-toxic over neuroblastoma cells. These findings revealed that high molecular mass oligomers of mortalin and Hsp70-1A preserved some of the fundamental functions of these proteins.

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.

Reactions of Ni(II) and Pd(II) precursors with S-benzyl-N-(ferrocenyl)methylenedithiocarbazate (HFedtc) led to the formation of heterobimetallic complexes of the type [MII(Fedtc)2] (M = Ni and Pd). The characterization of the compounds involved the determination of melting point, FTIR, UV–Vis, 1H NMR, elemental analysis and electrochemical experiments. Furthermore, the crystalline structures of HFedtc and [NiII(Fedtc)2] were determined by single crystal X-ray diffraction. The compounds were evaluated against the intracellular form of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity assays were assessed using LLC-MK2 cells. The results showed that the coordination of HFedtc to Ni(II) or Pd(II) decreases the in vitro trypanocidal activity while the cytotoxicity against LLC-MK2 cells does not change significantly. [PdII(Fedtc)2] showed the greater potential between the two complexes studied, showing an SI value of 8.9. However, this value is not better than that of the free ligand with an SI of 40, a similar value to that of the standard drug benznidazole (SI = 48). Additionally, molecular docking simulations were performed with Trypanosoma cruzi Old Yellow Enzyme (TcOYE), which predicted that HFedtc binds to the protein, almost parallel to the flavin mononucleotide (FMN) prosthetic group, while the [NiII(Fedtc)2] complex was docked into the enzyme binding site in a significantly different manner. In order to confirm the hypothetical interaction, in vitro experiments of fluorescence quenching and enzymatic activity were performed which indicated that, although HFedtc was not processed by the enzyme, it was able to act as a competitive inhibitor, blocking the hydride transfer from the FMN prosthetic group of the enzyme to the menadione substrate.

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain. However, the interaction is transient, creating a challenge for detailed analysis. We investigated whether it would be possible to gain further understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale is to increase the proximity between the interacting partners to promote their natural interaction and facilitate the characterization of the interaction. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the full-length Hsp70 or in the SBD alone, for instance a higher protective effect against aggregation and being a monomer. Substrate binding also appear to exceed that of SBD alone as revealed by a decreased binding to bis-ANS, a probe for hydrophobic patches. This hypothesis is supported by the structural model created by small angle X-ray scattering, suggesting that the lid subdomain (SBDα) is partially opened in the J-SBD. Collectively, our results suggest a model in which J-domain binding may shift the Hsp70 equilibrium towards the monomer state, exposing hydrophobic sites prone to substrate accommodation.

Made available in DSpace on 2019-10-04T12:42:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundacao Arthur Bernardes (FUNARBE - Program FUNARBE/FUNARPEX) The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (-50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated Delta N Delta S-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain. (C) 2019 Published by Elsevier B.V. Univ Fed Vicosa, Dept Bioquim & Biol Mol, Vicosa, MG, Brazil Univ Sao Paulo, Inst Quim Sao Carlos, Sao Carlos, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Genet Evolucao Microbiol & Imunol, Campinas, SP, Brazil Univ Fed Vicosa, Dept Vet, Vicosa, MG, Brazil Univ Estadual Campinas, Inst Biol, Dept Bioquim & Biol Tecidual, Campinas, SP, Brazil Univ Estadual Campinas, Fac Ciencias Farmaceut, Campinas, SP, Brazil Univ Estadual Campinas, Struct Genom Consortium, Av Dr Andre Tosello 550, Campinas, SP, Brazil Univ Oxford, Struct Genom Consortium, Old Rd Campus,Res Bldg,Roosevelt Dr, Oxford OX3 7DQ, England Univ Estadual Campinas, CBMEG, Ctr Mol Biol & Genet Engn, Campinas, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil CNPq: 485011/2012-3 CNPq: 420648/2016-0 CNPq: 471415/2013-8 CNPq: 303129/2015-8 FAPEMIG: CBB-APQ-01637-13 FAPEMIG: CBB-APQ-02556-15 FAPEMIG: RED-00140-16 FAPESP: 2011/23110-0 FAPESP: 2012/50161-8 FAPESP: 2014/07206-6 FAPESP: 2017/07335-9 FAPESP: 2012/00195-3 FAPESP: 13/50724-5 FAPESP: 2017/03489-1 CAPES: 001

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.

Mitochondrial Hsp70 (HSPA9, mtHsp70, mortalin) in conjunction with a complex set of other proteins is involved in the transport of polypeptides across the mitochondrial matrix. This observation allows us to hypothesize that HSPA9 might interact with membranes directly, similarly to other Hsp70s. Thus, we investigated whether human HSPA9 could also get inserted into lipid membranes. Human HSPA9 was incubated with liposomes made of lipids found within the mitochondrial membrane, such as 1', 3'-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-glycerol (CL), palmitoyl-oleoyl phosphocholine (POPC), palmitoyl-oleoyl phosphoserine (POPS), and palmitoyl-oleoyl phosphoethanolamine (POPE). HSPA9 displayed a predilection for CL and POPS, and low affinity for POPC and POPE, suggesting that the proteins have high specificity for negatively charged phospholipids. Then, liposomes were made with a composition resembling either the outer or inner mitochondrial membrane (OMM or IMM, respectively). We observed that HSPA9 has a higher affinity for IMM than OMM, which is consistent with the higher content of CL in the IMM. A comparison for the incorporation into POPS or CL liposomes by HSPA9 or HSPA1 indicated that both proteins behaved very similarly when exposed to CL liposomes, but differently with POPS liposomes, which was further corroborated by their susceptibility to proteinase K digestion after incorporation into liposomes. The measurement of thermodynamic parameters also showed that the interaction of both proteins with CL and POPS liposomes was different. Overall, our data showed that HSPA9 is prone to interact with membranes resembling the IMM that may be important for its role in the translocation of proteins into the mitochondria.

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.

Made available in DSpace on 2018-12-11T16:52:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Monoclonal antibody (mAb) delivery is gaining importance for local, systemic, and route-specific targeting. The mucus constitutes the main barrier for this type of delivery. In the present study, we aimed to develop a drug delivery platform by integrating mucus penetrating and mucoadhesive agents into a single system. Our hypothesis is that by combining these opposing functions, this system could have its properties modulated according to specific purposes. Self-assembly studies were conducted using three classes of building blocks: the protein drug bevacizumab (BVZ), mucus-penetrating polyanion dextran sulfate (DS), mucoadhesive polycations trimethylchitosan (TMC) and chitosan oligosaccharides (COS). We obtained two types of nanoparticles by manipulating supramolecular interactions between the components. Binary protein-polyanion (BVZ/DS) nanoparticles showed size of approximately 150 nm and a negative zeta potential. Ternary protein-polyanion-polycation (BVZ/DS/COS) nanoparticles were obtained using COS and exhibited 350 nm and a positive zeta potential. Assisted by calorimetric information, we demonstrated that building stable ternary nanoparticles carrying positive charges were not possible using the polycation TMC due to its thermodynamic constraints. Furthermore, spectroscopy analysis and CAM assay indicated that BVZ continued structurally and functionally stable after its incorporation into the nanoparticles. These two types of nanoparticles exhibited different behaviors when interacting with mucin, as shown by DLS and AFM studies. While the negatively charged particles promoted dispersion of the mucin network, suggesting a mucus penetrating effect of DS, the positively charged particles formed aggregates, probably caused by the mucoadhesive effect of COS. These results highlight the importance of understanding the role of supramolecular interactions, responsible for forming drug delivery systems containing complex molecules, such as proteins and polymers. School of Pharmaceutical Science São Paulo State University UNESP, Rodovia Araraquara/Jaú Km 1 Institute of Chemistry of Araraquara São Paulo State University UNESP Biophotonics Laboratory São Carlos Institute of Physics University of São Paulo (USP) EMBRAPA, Semi-Arid, Rodovia BE-428, Km 152, P.O. Box 23 EMBRAPA Instrumentation Rua XV de Novembro, 1.452, P.O. Box 741 Institute of Chemistry of São Carlos University of São Paulo USP School of Pharmaceutical Science São Paulo State University UNESP, Rodovia Araraquara/Jaú Km 1 Institute of Chemistry of Araraquara São Paulo State University UNESP

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.

Proteins participate in almost every cell physiological function, and to do so, they need to reach a state that allows its function by folding and/or exposing surfaces of interactions. Spontaneous folding in the cell is in general hindered by its crowded and viscous environment, which favors misfolding and nonspecific and deleterious self-interactions. To overcome this, cells have a system, in which Hsp70 and Hsp90 play a central role to aid protein folding and avoid misfolding. The topics of this review include the biophysical tools used for monitoring protein-ligand and protein-protein interactions and also some important results related to the study of molecular chaperones and heat shock proteins (Hsp), with a focus on the Hsp70/Hsp90 network. The biophysical tools and their use to probe the conformation and interaction of Hsp70 and Hsp90 are briefly reviewed.

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.

This paper presents an analysis of student perspectives in a public college of management, signatory of the Principles for Responsible Management Education (PRME) and demonstrates how these students are able to promote the 2030 Agenda of the Sustainable Development through their student organizations. Student organizations may be considered Communities of Practice (CoPs) since they are dynamic, interactive and not controlled by the institution. Thus, an exchange of knowledge and participants’ engagement occurs naturally through shared values and practices. The proposed research question is “What are the values and practices shared in student organizations that are interpreted as CoPs in a public college of management that has concrete actions and potential to promote the 2030 Agenda for Sustainable Development?” A questionnaire was sent to all of the members of the nine student organizations, and it contained six open-ended questions as well as profile questions. The answers to the open-ended questions were submitted to qualitative and quantitative content analysis. The results show that there is evidence that student organizations connect people by their beliefs, passions and shared values to achieve several goals, acting as CoPs, and have concrete actions and potential to promote the Sustainable Development Goals (SDGs).

For many students, education does not simply mean obtaining a university degree and, consequently, a good job; rather, there is also a need to attain personal satisfaction and a sense of responsibility for the community in which one lives. In the business school that we studied, we observed that learning not only occurs in the formal education program but also is complemented by operations in the nine existing student organizations, which constitute much of the school's hidden curriculum. This research entailed a survey with a qualitative approach. Analyzing the keyword answers of the students, 91% affirmed that they dealt with ethical concerns, corporate social responsibility and sustainability in student organizations. Based on the content of the other answers of the questionnaire, it can be concluded that students are creating their own learning content in student organizations in varied subjects to meet demands unmet by the formal curriculum. The student organizations are a fertile field for reflecting on and practicing Sustainable Development Goals (SDGs) and learning the Principles for Responsible Management Education (PRME). The students of the surveyed student organizations demonstrated a predisposition to social impact actions, thus ensuring their responsibility, ethics, interest in sustainability and awareness of society.

Alginate hydrogel improves anti-angiogenic bevacizumab activity in cancer therapy, Anti-vascular endothelial growth factor (anti-VEGF) therapy applied to solid tumors is a promising strategy, yet, the challenge to deliver these agents at high drug concentrations together with the maintenance of therapeutic doses locally, at the tumor site, minimizes its benefits. To overcome these obstacles, we propose the development of a bevacizumab-loaded alginate hydrogel by electrostatic interactions to design a delivery system for controlled and anti-angiogenic therapy under tumor microenvironrnental conditions. The tridimensional hydrogel structure produced provides drug stability and a system able to be introduced as a flowable solution, stablishing a depot after local administration. Biological performance by the chick embryo chorioallantoic membrane (CAM) assay indicated a pH-independent improved anti-angiogenic activity (similar to 50%) compared to commercial available anti-VEGF drug. Moreover, there was a considerable regression in tumor size when treated with this system. Immunohistochemistry highlighted a reduced number and disorganization of microscopic blood vessels resulting from applied therapy. These results suggest that the developed hydrogel is a promising approach to create an innovative delivery system.that offers the possibility to treat different solid tumors by intratumoral administration., Brazilian Fundação de Amparo e Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Additionally, this article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) Project PTDC/SAU-TOX/114549/2009 – FCOMP-01-0124-FEDER-016057, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038, info:eu-repo/semantics/publishedVersion