Solution structure of Plasmodium falciparum Hsp90 indicates a high flexible dimer.

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.

Discovery of small molecule inhibitors of Leishmania braziliensis Hsp90 chaperone.

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.

Structural and functional studies of the Leishmania braziliensis mitochondrial Hsp70: Similarities and dissimilarities to human orthologues.

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.

Heterotypic Coiled-Coil Formation is Essential for the Correct Assembly of the Septin Heterofilament.

Protein-protein interactions play a critical role in promoting the stability of protein quaternary structure and in the assembly of large macromolecular complexes. What drives the stabilization of such assemblies is a central question in biology. A limiting factor in fully understanding such systems is the transient nature of many complexes, making structural studies difficult. Septins comprise a conserved family of guanine nucleotide binding proteins that polymerize in the form of heterofilaments. In structural terms, they have a common organization: a central GTPase domain, an N-terminal domain, and a C-terminal domain; the latter is predicted to form a coiled coil. Currently, even for the best characterized human septin heterocomplex (SEPT2/SEPT6/SEPT7), the role of C-terminal domain is not fully established, and this is partly due to the absence of electron density for the C-terminal domains in the x-ray structure. Here we present results on the homo/heterotypical affinity for the C-terminal domains of human septins belonging to the SEPT6 and SEPT7 groups (SEPT6C/8C/10C/11C and SEPT7C, respectively) and provide clear evidence that this domain determines the preference for heterotypic interactions at one specific interface during the assembly of the heterofilament. This observation has wider implications where macromolecular assemblies are defined by coiled-coil protein interactions.

Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

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.

The C-terminal region of the human p23 chaperone modulates its structure and function.

The p23 protein is a chaperone widely involved in protein homeostasis, well known as an Hsp90 co-chaperone since it also controls the Hsp90 chaperone cycle. Human p23 includes a ß-sheet domain, responsible for interacting with Hsp90; and a charged C-terminal region whose function is not clear, but seems to be natively unfolded. p23 can undergo caspase-dependent proteolytic cleavage to form p19 (p231-142), which is involved in apoptosis, while p23 has anti-apoptotic activity. To better elucidate the function of the human p23 C-terminal region, we studied comparatively the full-length human p23 and three C-terminal truncation mutants: p231₋117; p231₋131 and p231₋142. Our data indicate that p23 and p19 have distinct characteristics, whereas the other two truncations behave similarly, with some differences to p23 and p19. We found that part of the C-terminal region can fold in an α-helix conformation and slightly contributes to p23 thermal-stability, suggesting that the C-terminal interacts with the ß-sheet domain. As a whole, our results suggest that the C-terminal region of p23 is critical for its structure-function relationship. A mechanism where the human p23 C-terminal region behaves as an activation/inhibition module for different p23 activities is proposed.

In-hospital mortality risk prediction after percutaneous coronary interventions: Validating and updating the Toronto score in Brazil.

OBJECTIVES: We aimed to assess the accuracy of the simple, contemporary and well-designed Toronto PCI mortality risk score in ICP-BR registry, the first Brazilian PCI multicenter registry with follow-up information. BACKGROUND: Estimating percutaneous coronary intervention (PCI) mortality risk by a clinical prediction model is imperative to help physicians, patients and family members make informed clinical decisions and optimize participation in the consent process, reducing anxiety and improving quality of care. At a healthcare system level, risk prediction scores are essential to measure and benchmark performance. METHODS: Between 2009 and 2013, a cohort of 4,806 patients from the ICP-BR registry, treated with PCI in eight tertiary referral medical centers, was included in the analysis. This population was compared to 10,694 patients of the derivation dataset from the Toronto study. To assess predictive performance, an update of the model was performed by three different methods, which were compared by discrimination, calculating the area under the receiver operating characteristic curve (AUC), and by calibration, assessed through Hosmer-Lemeshow (H-L) test and graphical analysis. RESULTS: Death occurred in 2.6% of patients in the ICP-BR registry and in 1.3% in the Toronto cohort. The median age was 64 and 63 years, 23.8 and 32.8% were female, 28.6 and 32.3% were diabetics, respectively. Through recalibration of intercept and slope (AUC = 0.8790; H-L P value = 0.3132), we achieved a well-calibrated and well-discriminative model. CONCLUSIONS: After updating to our dataset, we demonstrated that the Toronto PCI in-hospital mortality risk score performed well in Brazilian hospitals.

The regulation of the thermal stability and affinity of the HSPA5 (Grp78/BiP) by clients and nucleotides is modulated by domains coupling.

The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.

Type I Hsp40s/DnaJs aggregates exhibit features reminiscent of amyloidogenic structures.

A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and ß-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this ß-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar ß-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species in vivo.

Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi.

Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.

Gold(I) and Silver(I) Complexes Containing Hybrid Sulfonamide/Thiourea Ligands as Potential Leishmanicidal Agents.

Leishmaniasis is a group of parasitic diseases with the potential to infect more than 1 billion people; however, its treatment is still old and inadequate. In order to contribute to changing this view, this work consisted of the development of complexes derived from MI metal ions with thioureas, aiming to obtain potential leishmanicidal agents. The thiourea ligands (HLR) were obtained by reactions of p-toluenesulfohydrazide with R-isothiocyanates and were used in complexation reactions with AgI and AuI, leading to the formation of complexes of composition [M(HLR)2]X (M = Ag or Au; X = NO3- or Cl-). All compounds were characterized by FTIR, 1H NMR, UV-vis, emission spectroscopy and elemental analysis. Some representatives were additionally studied by ESI-MS and single-crystal XRD. Their properties were further analyzed by DFT calculations. Their cytotoxicity on Vero cells and the extracellular leishmanicidal activity on Leishmania infantum and Leishmania braziliensis cells were evaluated. Additionally, the interaction of the complexes with the Old Yellow enzyme of the L. braziliensis (LbOYE) was examined. The biological tests showed that some compounds present remarkable leishmanicidal activity, even higher than that of the standard drug Glucantime, with different selectivity for the two species of Leishmania. Finally, the interaction studies with LbOYE revealed that this enzyme could be one of their biological targets.

An Overview of Treatment Guidelines and Methods of Synthesis of Drugs Used in Leprosy Chemotherapy.

Leprosy is a Neglected Tropical Disease (NTDs) caused by Mycobacterium leprae (M. leprae). The treatment is considered effective, however, the high dose Multidrug Therapy (MDT) for a long period and its adverse effects result in the abandonment of the treatment by patients. Indeed, antimicrobial resistance is still an obstacle that must be overcome in the treatment of leprosy. In the present article, we reviewed the WHO guidelines for the chemotherapy of leprosy and the methods of synthesis of these drugs.

A Short Review of Antimalarial Compounds with Sulfonamide Moiety.

Malaria is a public health problem that causes thousands of deaths, primarily in children in African regions. Artemisinin-based combination therapies (ACTs) have helped to save thousands of lives; however, due to Plasmodium's resistance to available treatments, there is a need to search for new low-cost drugs that act through different mechanisms of action to contain this disease. This review shows that compounds with sulfonamide moiety, possibly, act as inhibitors of P. falciparum carbonic anhydrases, moreover, when linked to a variety of heterocycles potentiate the activities of these compounds and may be used in the design of new antimalarial drugs.

4-(1H-Pyrazol-1-yl) benzenesulfonamide derivatives: identifying new active antileishmanial structures for use against a neglected disease.

Leishmaniasis is a neglected disease responsible for about 56,000 deaths every year. Despite its importance, there are no effective, safe and proper treatments for leishmaniasis due to strain resistance and/or drug side-effects. In this work we report the synthesis, molecular modeling, cytotoxicity and the antileishmanial profile of a series of 4-(1H-pyrazol-1-yl)benzenesulfonamides. Our experimental data showed an active profile for some compounds against Leishmania infantum and Leishmania amazonensis. The profile of two compounds against L. infantum was similar to that of pentamidine, but with lower cytotoxicity. Molecular modeling evaluation indicated that changes in electronic regions, orientation as well as lipophilicity of the derivatives were areas to improve the interaction with the parasitic target. Overall the compounds represent feasible prototypes for designing new molecules against L. infantum and L. amazonensis.

Corrigendum: Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania Amazonensis Developmental Cycle and Population Proliferation.

[This corrects the article DOI: 10.3389/fcell.2021.713415.].

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes.

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.

Central domain deletions affect the SAXS solution structure and function of yeast Hsp40 proteins Sis1 and Ydj1.

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.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70.

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes.

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.

Purification and characterization of a novel and conserved TPR-domain protein that binds both Hsp90 and Hsp70 and is expressed in all developmental stages of Leishmania major.

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.