Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants.

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The 'T' component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

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.

Crystal structure of a novel xylose isomerase from Streptomyces sp. F-1 revealed the presence of unique features that differ from conventional classes.

BACKGROUND: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. METHODS: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain. RESULTS: We showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. CONCLUSIONS: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. GENERAL SIGNIFICANCE: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass.

Thermodynamic analysis of interactions of the Hsp90 with adenosine nucleotides: A comparative perspective.

Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp90s are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp90s 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 Hsp90s from different organisms (human α and ß 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.

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

Insights on the structure of amyloid fibrils from site-directed mutagenesis.

To test the hypothesis that the ability to form ordered beta-rich amyloid fibers with identical structures is a generic property of proteins we present a study on the overall structures of fibers formed by apomyoglobin mutants that either stabilize or destabilize the native state or the intermediate. Our results indicate that, at least at the macroscopic level, ordered beta-rich amyloid fibers have similar structures.

Molecular dynamics and circular dichroism studies of human and rat C-peptides.

Proinsulin C-peptide has been recently described as an endogenous peptide hormone, responsible for important physiological functions others than its role in proinsulin processing. Accumulating evidences that C-peptide exerts beneficial effects in the treatment of long term complications of patients with type 1 diabetes mellitus indicate that this molecule may be administered together with insulin in future therapies. Despite its clear pharmacological interest, the secondary and three-dimensional (3D) structures of human C-peptide are still points of controversy. In the present work we report molecular dynamics (MD) simulations of human, rat I and rat II C-peptides. A common experimental strategy applied to all peptides consisted of homology building followed by multinanosecond MD simulations in vacuum and water. Circular dichroism (CD) experiments of each peptide in the absence and presence of 2,2,2-trifluoroethanol (TFE) were performed to support validation of the theoretical models. A multiple sequence alignment of 23 known mammalian C-peptides was constructed to identify significant conserved sites that would be important for the maintenance of secondary and tertiary structures. The analysis of the molecular dynamics trajectories for the human, rat I and rat II molecules have shown quite different general behavior, being the human C-peptide more flexible than the two others. Human and rat C-peptides exhibit very stable turn-like structures at the middle and C-terminal regions, which have been described as potential active sites of C-peptides. Human C-peptide also presented a short alpha-helix throughout the MD, which was not found in the rat molecules. CD data is in very good agreement with the MD results and both methods were able to identify a greater structural stability and potential in rat C-peptides when compared to the human C-peptide. The simulation results are discussed and validated in the light of multiple sequence alignment, recent experimental data from the literature and our own CD experiments.