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Chaperones belonging to the small heat shock protein (sHSP) family are ubiquitous and exhibit elevated expression under stresses conditions to protect proteins against aggregation, thereby contributing to the stress tolerance of the organism. Tropical plants are constantly exposed to high temperatures, and the mechanisms by which these plants tolerate heat stress are of foremost importance to basic science as well as applied agrobiotechnology. Therefore, this study aims to characterize sHSPs from different organelles from sugarcane, an important crop that is associated with sugar and bioenergy production. An expression sequence tag database of sugarcane was searched, and sHsp genes of mitochondrial and chloroplast organelles were selected and cloned. The proteins were expressed in Escherichia coli and isolated and purified by two chromatographic steps with high purity as single species. Circular dichroism and fluorescence spectroscopy showed that both proteins were purified in their folded states with a predominant β-sheet secondary structure. Determination of the molecular weight, diffusion coefficient and Stokes radius parameters showed that both chaperones form large spherical-like oligomers in solution. The two sHSPs had different oligomeric states and substrate specificities. The mitochondrial sHSP was a 20-mer with ability to protect model substrates that differ from that of the 16-meric sHSP from chloroplasts. These results indicate that both sHSPs are key agents to protect against stress confirming the importance of the great diversity of sHSP chaperones in plants for homeostasis maintenance. Moreover, to our knowledge, this is the first report about small HSPs from sugarcane organelles. [ABSTRACT FROM AUTHOR]

Abstract: In the cell, proteins interact within a network in which a small number of proteins are highly connected nodes or hubs. A disturbance in the hub proteins usually has a higher impact on the cell physiology than a disturbance in poorly connected nodes. In eukaryotes, the cytosolic Hsp90 is considered to be a hub protein as it interacts with molecular chaperones and co-chaperones, and has key regulatory proteins as clients, such as transcriptional factors, protein kinases and hormone receptors. The large number of Hsp90 partners suggests that Hsp90 is involved in very important functions, such as signaling, proteostasis and epigenetics. Some of these functions are dysregulated in cancer, making Hsp90 a potential target for therapeutics. The number of Hsp90 interactors appears to be so large that a precise answer to the question of how many proteins interact with this chaperone has no definitive answer yet, not even if the question refers to specific Hsp90s as one of the human cytosolic forms. Here we review the major chaperones and co-chaperones that interact with cytosolic Hsp90s, highlighting the latest findings regarding client proteins and the role that posttranslational modifications have on the function and interactions of these molecular chaperones. This article is part of a Special Issue entitled: Proteomics: The clinical link. [Copyright &y& Elsevier]

Abstract: Protein misfolding is stimulated by stress, such as heat, and heat shock proteins (Hsps) are the first line of defense against these undesirable situations. Plants, which are naturally sessile, are perhaps more exposed to stress factors than some other organisms, and consequently, the role of Hsps is crucial to maintain homeostasis. Hsp90, because of its key role in infection and other stresses, is targeted in therapies that improve plant production by increasing resistance to both biotic and abiotic stress. In addition, Hsp90 is a primary factor in the maintenance of homeostasis in plants. Therefore, we cloned and purified Hsp90 from Citrus sinensis (sweet orange). Recombinant C. sinensis Hsp90 (rCsHsp90) was produced and measured by circular dichroism (CD), intrinsic fluorescence spectroscopy and dynamic light scattering. rCsHsp90 formed a dimer in solution with a Stokes radius of approximately 62Å. In addition, it was resistant to thermal unfolding, was able to protect citrate synthase from aggregation, and Western blot analysis demonstrated that CsHsp90 was constitutively expressed in C. sinensis cells. Our analysis indicated that CsHsp90 is conformationally similar to that of yeast Hsp90, for which structural information is available. Therefore, we showed that C. sinensis expresses an Hsp90 chaperone that has a conformation and function similar to other Hsp90s. [Copyright &y& Elsevier]

Abstract: The natural N- and C-termini, i.e., the given order of secondary structure segments, are critical for protein folding and stability, as shown by several studies using circularly permuted proteins, mutants that have their N- and C-termini linked and are then digested at another site to create new termini. A previous work showed that circularly permuted mutants of sperm whale myoglobin (Mb) are functional, have native-like folding and bind heme, but are less stable than the wild-type protein and aggregate. The ability of wild-type myoglobin to form amyloid fibrils has been established recently, and because circularly permuted mutations are destabilizing, we asked whether these permutations would also affect the rate of amyloid fibril formation. Our investigations revealed that, indeed, the circularly permuted mutants formed cytotoxic fibrils at a rate higher than that of the wild-type. To further investigate the role of the C-terminus in the overall stability of the protein, we investigated two C-terminally deleted mutant, Mb1–123 and Mb1–99, and found that Mb1–123 formed cytotoxic fibrils at a higher rate than that of the wild-type while Mb1–99 formed cytotoxic fibrils at a similar rate than that of the wild-type. Collectively, our findings show that the native position of both the N-and C-termini is important for the precise structural architecture of myoglobin. [Copyright &y& Elsevier]

Abstract: Small heat shock proteins (sHsp) constitute an important chaperone family linked to conformational diseases. In plants, sHsps prevent protein aggregation by acting as thermosensors and to enhance cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I sHsps in sugarcane. They exist as dodecamers at 20 °C and have distinct substrate specificities. Therefore, they are useful models to study how class I SHsps work. Here we present data on the effects of heat on the oligomerization and chaperone activity of SsHsp17.2 and SsHsp17.9. Using several biophysical and biochemical probes, we show that the effects of heat are completely reversible, an important property for proteins that act at heat shock temperatures. SsHsp17.2 and SsHsp17.9 dodecamers dissociated to dimers at temperatures ranging from 40 to 45 °C and this dissociation was followed by enhanced chaperone activity. We conclude that high temperature affects the oligomeric state of these chaperones, resulting in enhanced chaperone activity. [Copyright &y& Elsevier]

Abstract: Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed. [Copyright &y& Elsevier]

Abstract: When cells are submitted to an increase in temperature, heat shock proteins (Hsp) are synthesized to help heat stress resistance. Small Hsps, which are diverse and abundant in plants, have the major function of preventing irreversible protein aggregation. The diversity of small Hsps in plants is intriguing and characterization of their chaperone activity is important to understand plant tolerance to heat stress. A previous study showed that small Hsps, mainly represented by class I (cytosolic), correspond to about 5% of all sugarcane Expressed Sequencing Tags belonging to the molecular chaperone category. Here, we present biochemical and biophysical characterization of two sugarcane small Hsps from class I, which were named SsHsp17.2 and SsHsp17.9 according to their monomer molecular mass of 17.2 and 17.9kDa, respectively. The recombinant proteins have identity of about 75% to each other and similar structural characteristics. However, their stability and their chaperone activity were not equivalent: SsHsp17.9 was more efficient in protecting citrate synthase and malate dehydrogenase from aggregation whereas SsHsp17.2 was more efficient in protecting luciferase from aggregation. There is only one region, which is located at the N-terminus, of low homology between these two proteins. Based on that and on previous works pointing to multiple sites, mainly at the N-terminus, involved with substrate specificity in small Hsps, we suggest that this specific region is one of these sites. In addition, this is the first report on the chaperone activity of sugarcane small Hsps. [Copyright &y& Elsevier]

Abstract: Hsp70 alternates between an ATP-bound state in which the affinity for substrate is low and an ADP-bound state in which the affinity for substrate is high, as a result Hsp70 assists the protein folding process through nucleotide-controlled cycles of substrate binding and release. In this work, we describe the cloning and purification of the human 70-kDa heat shock cognate protein, Hsc70, and the use of circular dichroism, intrinsic emission fluorescence, and isothermal titration calorimetry to characterize conformational changes induced by ADP and ATP binding. Binding of either ADP or ATP were not accompanied by a net change in secondary structure suggesting that the conformational rearrangement caused by nucleotide binding is localized. MgADP or MgATP had a greater effect in the stability at stress temperatures than ADP or ATP did. Isothermal titration calorimetry data pointed out that Hsc70 had a lower affinity for ATP (K D =710nM) than for ADP (K D =260nM). [Copyright &y& Elsevier]

We report here on the stability and folding of the 91 residue α-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC1–91F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC1–91F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed. [Copyright &y& Elsevier]

Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins. [Copyright &y& Elsevier]

Examines putative interhelix ion pairs involved in the stability of sperm whale myoglobin. Single mutations of sperm whale myoglobin; Favorable contribution of the electrostatic interactions to myoglobin stability at a certain pH level; Factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain.

Investigates the effects of mutations at helix pairing sites on the stability of the apomyoglobin intermediate I. Truncate hydrophobic side chains; Specificity of hydrophobic burial to native helix packing sites; Role of hydrophobic burial in the formation of the cooperatively folded intermediate.

The emergence of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a great threat to global health. ORF9b, an important accessory protein of SARS-CoV-2, plays a critical role in the viral host interaction, targeting TOM70, a member of the mitochondrial translocase of the outer membrane complex. The assembly between ORF9b and TOM70 is implicated in disrupting mitochondrial antiviral signaling, leading to immune evasion. We describe the expression, purification, and characterization of ORF9b alone or coexpressed with the cytosolic domain of human TOM70 in E. coli. ORF9b has 97 residues and was purified as a homodimer with an molecular mass of 22 kDa as determined by SEC-MALS. Circular dichroism experiments showed that Orf9b alone exhibits a random conformation. The ORF9b-TOM70 complex characterized by CD and differential scanning calorimetry showed that the complex is folded and more thermally stable than free TOM70, indicating strong binding. Importantly, protein–protein interaction assays demonstrated that full-length human Hsp90 is capable of binding to free TOM70 but not to the ORF9b-TOM70 complex. To narrow down the nature of this inhibition, the isolated C-terminal domain of Hsp90 was also tested. These results were used to build a model of the mechanism of inhibition, in which ORF9b efficiently targets two sites of interaction between TOM70 and Hsp90. The findings showed that ORF9b complexed with TOM70 prevents the interaction with Hsp90, and this is one major explanation for SARS-CoV-2 evasion of host innate immunity via the inhibition of the interferon activation pathway. [Display omitted] • Free ORF9b was purified as a homodimer. • ORF9b binds TOM70 as a monomer. • ORF9b-TOM70 complex is folded and more thermally stable than free TOM70. • Binding of SARS-CoV-2 ORF9b to TOM70 prevents its interaction with HSP90. [ABSTRACT FROM AUTHOR]

The imbalance in metal homeostasis can be associated with several human diseases, and exposure to increasing concentrations of metals promotes cell stress and toxicity. Therefore, understanding the cytotoxic effect of metal imbalance is important to unravel the biochemical mechanism of homeostasis and the action of potential protective proteins against metal toxicity. Several studies, including gene deletion in yeast, provide evidence indicating the possible indirect involvement of cochaperones from the Hsp40/DNAJA family in metal homeostasis, possibly through modulating the activity of Hsp 70.This work first investigated the effect of zinc and copper on the conformation and function of the human Hsp40 cochaperone DNAJA1, a zinc-binding protein. DNAJA1 was capable to complement the phenotype of a yeast strain deleted of the ydj1 gene, which was more sensitive to the presence of zinc and copper than the wild-type strain. To gain further insight about the role of the DNAJA family in metal binding, the recombinant human DNAJA1 protein was studied. Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone, i.e., to protect other proteins from aggregation. The reintroduction of zinc restored the native properties of DNAJA1 and, surprisingly, the addition of copper partially restored the native properties. [Display omitted] • Human DNAJA1 is capable to complement the phenotype of a yeast strain deleted of the ydj1 gene. • Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone. • The reintroduction of either zinc restored the native properties of DNAJA1. • The reintroduction of either copper partially restored the native properties of DNAJA1. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease caused by protozoans of the Leishmania genus, against which no effective treatment or control is available. Like other eukaryotes, parasite telomeres are maintained by telomerase, a ribonucleoprotein complex vital for genome stability. Its protein component, TERT (telomerase reverse transcriptase), presents four structural and functional domains, with the TEN (Telomerase N-terminal) and TRBD (Telomerase RNA-binding) located at its N-terminal. The enzyme also contains an RNA component that carries the template copied by the TERT during telomere elongation. Here, we show that the tertiary structure of Leishmania major TERT (LmTERT) is conserved compared to other eukaryotes. However, the LmTERT N-terminal (LmTERT-NT) portion shows structural changes not detected in the entire protein, mainly in the TEN domain. Besides the disordered elements, the TEN gains two long β-sheets but preserves the GQ motif and the residues in β-sheet 5 that interact with the TRAP motif. In both structures, a linker flanks the TEN and TRBD. The TRBD is partially conserved in both structures and contains the canonical QFP and T motifs, invariant residues, and the putative CP and two trypanosomatid-specific motifs (TSM) besides genus-specific amino acid substitutions. Despite the structural changes, the recombinant LmTERT-NT preserves a hydrophobic cavity that binds specifically and in the picomolar range to the telomeric G-rich DNA and the TER 5′ end region. Thus, LmTERT-NT shares the canonical structural domains and motifs and is biochemically active. We discuss the importance of the TERT N-terminal region in the parasite's telomerase catalysis. [Display omitted] • AlphaFold2 generated the complete LmTERT and the LmTERT-NT structures. • The structural analysis of LmTERT and LmTERT-NT revealed they preserved the canonical domains and motifs. • Recombinant LmTERT-NT was expressed and is biologically active. • CD and fluorescence spectroscopy confirmed the protein's structural integrity. • Recombinant LmTERT-NT binds the telomeric DNA and the telomerase RNA component. [ABSTRACT FROM AUTHOR]

Background Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. Results In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Conclusion Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. General significance To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α. [ABSTRACT FROM AUTHOR]

Heat shock protein 40 (Hsp40) co-chaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by Hsp40s and a common mechanism for polypeptide transfer to Hsp70. [Copyright &y& Elsevier]

Summary: Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5′ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. [Copyright &y& Elsevier]

SGTs (small glutamine-rich TPR-containing proteins) are dimeric proteins that belong to the class of co-chaperones characterized by the presence of TPR domains (containing tetratricopeptide repeats). Human (SGTA) and yeast (Sgt2) SGTs are characterized by three distinct domains: an N-terminal dimerization domain, a central TPR-domain important for binding to other proteins (chaperones included) and a C-terminal domain involved in hydrophobic interactions. Both these SGTs are involved in the cellular PQC (protein quality control) system, as they interact with chaperones and have functions that aid stress recovery. However, there are differences between them, such as structural features and binding specificities, that could be better understood if other orthologous proteins were studied. Therefore, we produced and characterized a putative SGT protein, designated AaSGT, from the mosquito Aedes aegypti , which is a vector of several diseases, such as dengue and Zika. The protein was produced as a folded dimer which was stable up to 40 °C and was capable of binding to AaHsp90 and fully protecting a model protein, α-synuclein, from aggregation. The conformation of AaSGT was investigated by biophysical tools and small angle X-ray scattering, which showed that the protein had an elongated conformation and that its C-terminal domain was mainly disordered. The results with a C-terminal deletion mutant supported these observations. Altogether, these results are consistent with those from other functional SGT proteins and add to the understanding of the PQC system in Aedes aegypti , an important aim that may help to develop inhibitory strategies against this vector of neglected diseases. [Display omitted] • AaSGT provides phenotype complementation in yeast using a heat sensitivity assay. • AaSGT is capable of fully protecting α-Synuclein from aggregation. • AaSGT has an elongated conformation with a disordered C-terminus. [ABSTRACT FROM AUTHOR]

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. Image 1 • A novel TPR-domain protein (LmTPR) was identified in Leishmania major. • LmTPR is conserved in all Leishmania species analyzed and is expressed in all developmental stages of Leishmania major. • LmTPR binds both Hsp90 and Hsp70. [ABSTRACT FROM AUTHOR]

Cellular proteostasis is maintained by a system consisting of molecular chaperones, heat shock proteins (Hsps) and proteins involved with degradation. Among the proteins that play important roles in the function of this system is Hsp90, which acts as a node of this network, interacting with at least 10% of the proteome. Hsp90 is ATP-dependent, participates in critical cell events and protein maturation and interacts with large numbers of co-chaperones. The study of Hsp90 orthologs is justified by their differences in ATPase activity levels and conformational changes caused by Hsp90 interaction with nucleotides. This study reports the characterization of Hsp90 from Aedes aegypti , a vector of several diseases in many regions of the planet. Aedes aegypti Hsp90, AaHsp90, was cloned, purified and characterized for its ATPase and chaperone activities and structural conformation. These parameters indicate that it has the characteristics of eukaryotic Hsp90s and resembles orthologs from yeast rather than from human. Finally, constitutive and increased stress expression in Aedes cells was confirmed. Taken together, the results presented here help to understand the relationship between structure and function in the Hsp90 family and have strong potential to form the basis for studies on the network of chaperone and Hsps in Aedes. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor , a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated. Graphical abstract Unlabelled Image Highlights • Sorghum bicolor Hop (SbHop) is a mainly alpha-helical monomer. • The conserved SbHop fold provides the elongated protein with a high flexibility. • A conserved carboxylate clamp confers high affinity binding to Hsp90. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (c G MP-specific phosphodiesterases, a denylyl cyclases and F hlA) domain, four PAS ( P er- A rnt- S im) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a Xac Δ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H 2 O 2 . Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule ( K 1 ) approximately 5 × greater than the dissociation constant for the binding of the second GTP molecule ( K 2 ). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo . [ABSTRACT FROM AUTHOR]

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 (p23 1–142 ), 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: p23 1–117 ; p23 1–131 and p23 1–142 . 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. [ABSTRACT FROM AUTHOR]

Abstract: Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. Biological significance: Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics. [Copyright &y& Elsevier]

Abstract: Hsp90s are involved in several cellular processes, such as signaling, proteostasis, epigenetics, differentiation and stress defense. Although Hsp90s from different organisms are highly similar, they usually have small variations in conformation and function. Thus, the characterization of different Hsp90s is important to gain insight into the structure–function relationship that makes these chaperones key regulators in protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from sugarcane and its comparison with Hsp90s from other plants. Previous expressed sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the recombinant protein was purified and its conformation and function characterized. The structural conformation of Hsp90 was assessed by chemical cross-linking and hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays, which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong chaperone activity. [Copyright &y& Elsevier]

Abstract: The Clp/Hsp100 AAA+ chaperone family is involved in recovering aggregated proteins and little is known about other orthologs of the well studied ClpB from Escherichia coli and Hsp104 from Saccharomyces cerevisiae. Plant Hsp101 is a good model for understanding the relationship between the structure and function of Hsp100 proteins and to investigate the role of these chaperones in disaggregation processes. Here, we present the cloning and purification of a sugarcane ortholog, SHsp101, which is expressed in sugarcane cells and is a folded hexamer that is capable of binding nucleotides. Thus SHsp101 has the structural and functional characteristics of the Clp/Hsp100 AAA+ family. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. [Copyright &y& Elsevier]

Abstract: Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37°C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65°C) and exhibit α-helix content from ∼30% (XACb003) to ∼87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high α-helix content, and without ATP-binding or ATP-hydrolyzing activity. [Copyright &y& Elsevier]

Abstract: The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K d 0.29±0.12μM and 0.58±0.14μM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species. [Copyright &y& Elsevier]

Abstract: The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase. [Copyright &y& Elsevier]

Abstract: GrpE acts as a nucleotide exchange factor for the Hsp70 chaperone system. Only one GrpE isoform is present in Escherichia coli, but for reasons not yet well understood, two GrpE isoforms have been found in mammalian mitochondria.Therefore, studies aimed at evaluating the physico-chemical characteristics of these proteins are important for the comprehension of the function of the Hsp70 chaperone system in different organisms. Here we report biophysical studies on human mitochondrial GrpE isoform 2. Small angle X-ray scattering measurements of human GrpE isoform 2 showed that this protein has a quaternary structure which is similar to those of human GrpE isoform 1 and E. coli GrpE: a dimer with a cruciform elongated shape. However, mitochondrial isoforms differed from each other regarding chemical and thermal denaturation profiles. This fact, combined with results of distinct expression patterns previously reported, point out that these proteins may have different response to external stimuli. Our results also indicate that human GrpE isoform 2 is more similar to the GrpE from E. coli than to human GrpE isoform 1. These results are relevant because differences in the conformation of Hsp70 co-chaperones are considered to be one of the reasons for functional diversity of this system. [Copyright &y& Elsevier]

Abstract: In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world’s second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65Ǻ resolution. The MtCS structure is similar to other CS structures, presenting β–α–β sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event. [Copyright &y& Elsevier]

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6×His-Ki-1/57(122–413) and 6×His-Ki-1/57(264–413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein β subunit as template. Our model suggests the family-typical seven-bladed β-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57. [ABSTRACT FROM AUTHOR]

Proteolytic cleavage of the amyloid precursor protein (APP) by β and γ-secretases gives rise to the β-amyloid peptide, considered to be a causal factor in Alzheimer''s disease. Conversely, the soluble extracellular domain of APP (sAPPα), released upon its cleavage by α-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPα have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPα (sAPPα695 and sAPPα770) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPα, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPα isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPα forms a 2:1 complex with heparin. A conformational model for the sAPPα:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed. [Copyright &y& Elsevier]

Abstract: The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins. [Copyright &y& Elsevier]

The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK. [ABSTRACT FROM AUTHOR]

As molecular biology has developed it has become possible to abundantly produce heterologous proteins in bacteria and to design serial amino acid substitutions for the generation of modified proteins, an approach also known as protein engineering. Sperm whale myoglobin, a protein of broad interest, has been cloned for several years now and a large collection of mutants has been produced. The presence of heme stabilizes the protein, which is recovered soluble from the bacterial pellet, and most purification protocols take advantage of this property for myoglobin purification directly from the pellet. However, recovery from the column resin is poor with these methods making them expensive and the procedure for removing heme is laborious and drastic when the apo form of Mb is required. In the case of proteins with severe mutations, which bind heme weakly or do not bind it at all, such methods cannot be employed without massive loss of productivity. Here, we describe a modified method, which is both low cost and rapid, for the purification of the soluble apo form of Mb from Escherichia coli inclusion bodies. Biophysical characterization of the protein after purification shows that the purified apoMb retains its native conformation and is soluble. This modified method is also used for the purification of a non-heme-binding apoMb mutant, demonstrating its efficiency when dealing with drastic mutations. [Copyright &y& Elsevier]

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. [Display omitted] • R2TP, R2T, and R2P chaperone complexes are identified and characterized • RPAP3 and RPAP3-PIH1D1 are highly dynamic proteins • RPAP3 and PIH1D1 do not influence RUVBL1/2 ATPase activity • RPAP3 N-domain binds proteins enriched in helical domains R2TP is a conserved chaperone complex that is required for quaternary structure formation. Seraphim et al. perform detailed structural and biochemical analyses to understand the dynamics and mechanism of function of this complex. This work provides important insights into how a chaperone involved with complex assembly might work. [ABSTRACT FROM AUTHOR]

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major , one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitr o. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance. [ABSTRACT FROM AUTHOR]

α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease. Unlabelled Image • Impact of lipid size on α-syn structuring, LUVs vs lipid-bilayer nanodiscs encapsulated by DIBMA. • DIBMALPs are suitable nanoscale membrane mimics for studying αsyn secondary-structure. • In DIBMALPS folding was independent of L/P ratio, in contrast with LUVs. • New and promising application of polymer-encapsulated lipid-bilayer nanodiscs. • Human, elephant and whale αsyn were compared for α-helix formation in the presence of lipids. [ABSTRACT FROM AUTHOR]

• SbCHIP is a non-spherical symmetric dimer in solution. • SbCHIP forms complexes with Hsp70 and Hsp90 from plant and interacts with E2 enzymes UbcH5 and UbcH13. • SbCHIP is able of autoubiquitination, but in the presence of substrate SbCHIP ubiquitinates Hsp70, Hsp90 and luciferase. The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants. [ABSTRACT FROM AUTHOR]

Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases. Unlabelled Image [ABSTRACT FROM AUTHOR]

The phenomenon of surface plasmon resonance (SPR) through optical sensors was developed from initial studies involving excitation of surface plasmons on metallic substrates. From the beginning, these optical systems have attracted increasing interest for application in different areas, ranging from physics, chemistry, and materials science to biology. Although numerous applications have been explored, the use of SPR in the development of biosensors is by far the most prominent. This review provides a brief account of fundamental aspects related to the recent applications of SPR as a tool for the development of new clinical diagnosis methods. The applications of SPR biosensors were illustrated through recent studies published in the field of neglected tropical diseases, with an emphasis on the contributions achieved in visceral leishmaniasis. It was possible to demonstrate the real benefits and the difficulties that the SPR biosensors have encountered in this important and complex system. Finally, future trends in the use of nanomaterials for the development of SPR-based portable devices for application to neglected tropical diseases have been demonstrated. Image 1 • The SPR technique has been used for diagnostic purposes and for the elucidation of biochemical events. • Viability of SPR-based biosensor for the development of new clinical diagnosis methods. • SPR immunosensors showed higher sensitivity for the detection of leishmaniasis antibodies than the ELISA assay. •. The binding affinity values demonstrated high viability of the use of recombinant proteins in control programs. • Perspectives in using nanomaterials and portable device in SPR-based biosensors.. [ABSTRACT FROM AUTHOR]

Summary Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment. [ABSTRACT FROM AUTHOR]