Thiago V. Seraphim

A draft of the human septin interactome.

Background Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings Here, we performed yeast two-hybrid screens with human septins 1–10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the “group rule”, i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Low Resolution Structural Studies Indicate that the Activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis Has an Elongated Shape Which Allows Its Interaction with Both N- and M-Domains of Hsp90

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

Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

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

Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities

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

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.

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 β-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 β-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.

The Interaction Networks of Hsp70 and Hsp90 in the Plasmodium and Leishmania Parasites

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

Insights on the structural dynamics of Leishmania braziliensis Hsp90 molecular chaperone by small angle X-ray scattering

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

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.