Structural characterization of the human DjC20/HscB cochaperone in solution.

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.

Structural, thermodynamic and functional studies of human 71 kDa heat shock cognate protein (HSPA8/hHsc70).

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.

New insights on human Hsp70-escort protein 1: Chaperone activity, interaction with liposomes, cellular localizations and HSPA's self-assemblies remodeling.

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.

Human HSPA9 (mtHsp70, mortalin) interacts with lipid bilayers containing cardiolipin, a major component of the inner mitochondrial membrane.

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.

Interaction of HSPA5 (Grp78, BIP) with negatively charged phospholipid membranes via oligomerization involving the N-terminal end domain.

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.

Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice.

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.

Structural studies of Old Yellow Enzyme of Leishmania braziliensis in solution.

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/ß-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.