Protein and Peptide Letters - Volume 18, Issue 2, 2011

Molecular chaperones are a class of proteins that aid in the folding of other proteins. They are also referred to as heat-shock proteins (HSP) because they were first described as proteins induced during thermal stress. Although the term molecular chaperone was used first by Laskey and co-workers (Nature 275, 416-420, 1978) to describe the function of nucleoplasmin, a protein that promotes the correct oligomerization of histones by preventing their aggregation during assembly in chromatin, the concept of molecular chaperones may be better described by a statement from a paper published by Hendrick and Hartl (Annu. Rev. Biochem. 62, 349-84, 1993): “we define a molecular chaperone as a protein that binds to and stabilizes an otherwise unstable conformer of another protein, facilitates its correct fate in vivo: be it in folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between active/inactive conformations”. Shortly after their discovery, we learned that chaperones are expressed constitutively and assist in a diverse array of cellular processes that continually challenge cellular protein homeostasis and require protein quality control systems. In this context, molecular chaperones facilitate cellular protein homeostasis by promoting protein folding and degradation. In fact, many proteins become unstable at high temperatures and the heat shock response acts as a protective cellular mechanism against stress-induced protein damage. Failure to reach or maintain the correctly folded structure can have serious consequences and can lead to aggregation, which is often associated with the assembly of the misfolded protein into fibrillar aggregates. These aggregates are commonly known as amyloid fibrils and are associated with more than 40 pathological disorders in humans. Additionally, molecular chaperones are also responsible for maintaining the folding, stability and function of many proteins involved in signal-transduction pathways, cell-cycle regulation and apoptosis. Therefore, chaperones are considered to be stabilizers of the cancer phenotype. This special issue aims to provide current review articles about molecular chaperones and their role in protein quality control. The review by Tiroli-Cepeda and Ramos gives a general view of protein homeostasis as well as the structure and function of the main chaperone families. The review by Anderson et al. centers on protein disorders caused by defects on tRNA charging or abnormal protein degradation, ribosomopathies and chaperonopathies. Fan and Young give an overview of the important role of molecular chaperones on protein translocation into the mitochondria, which is critical for this organelle to maintain proper functions. Da Silva and Borges center their review on Hsp70, covering the structural and functional aspects of this chaperone machinery. Because chaperones are ubiquitous and essential for the life of all species, a greater understanding of the role of these chaperones in protein homeostasis in pathogens will aid in the fight against microorganisms. The review by Shonhai et al. describes molecular chaperones from protozoan parasites and their relation to human health, and the review by Fattori et al. describes the important role of chaperones in the secretion of virulence factors into host cells. Finally, Monteiro and Cano present the role of SIRT1 in protein homeostasis. SIRT1 has been implicated in aging and caloric restriction. Because this protein interacts with heat shock factors, the heat shock response appears to be even more relevant than previously thought as it is intrinsically involved in many aspects of human health. As seen from the aforementioned remarkable abilities of chaperones, these proteins are potential drug targets. Treatments for conformational diseases caused by protein misfolding may be achieved by enhancing the function of chaperones. On the other hand, anticancer therapies may be developed by inhibiting chaperones to significantly weaken a cancer cell, causing regression of tumor growth. It remains to be seen whether therapies involving molecular chaperones will be successful in reverting misfolded protein diseases. However, a better understanding of the mechanism of action of molecular chaperones will truly provide insight into cellular function under normal and stressed conditions.

Cells require a protein quality control (PQC) system to obtain a correct balance between folding and the degradation of incorrectly folded or misfolded proteins. This system maintains protein homeostasis and is essential for life. Key components of the PQC are molecular chaperones, which compose a ubiquitous class of proteins that mediate protein quality control by aiding in both the correct folding of proteins and the elimination of proteins that are misfolded due to cellular stress or mutation. Recent studies showed that protein homeostasis has an important role in nutrition and aging, increasing the relevance of the heat shock response to human health. This review summarizes our current knowledge of the molecular chaperone system and its role in protein homeostasis.

The great diversity of structural conformations available to proteins allows this class of molecules to carry out the vast majority of biochemical functions in the cell. In order to function adequately, proteins must be synthesized, folded/assembled and degraded with great temporal and spatial accuracy. Precise coordination of multiple processes, including ribosome assembly and movement along mRNA, charging and recycling of tRNAs, recruitment and action of molecular chaperones, and tight control of the degradation machinery is essential to create and maintain a stable proteome. It has become recently evident that even slight errors in any of these processes may lead to disease states. Accordingly, increasing numbers of human diseases have been identified that are due to mutations in genes encoding proteins involved in this so-called “protein quality control”. Since these processes are essential for the production and maintenance of the entire proteome of the cell, the deleterious effects of these mutations often extend far beyond the faulty gene. This review provides an overview of human disorders caused by defects in mechanisms underlying protein biogenesis and stability.

The great majority of mitochondrial proteins are synthesized by cytosolic ribosomes and then imported into the organelle post-translationally. The translocase of the outer membrane (TOM) is a proteinaceous machinery that contains surface receptors for preprotein recognition and also serves as the main entry gateway into mitochondria. Mitochondrial targeting requires various cytosolic factors, in particular the molecular chaperones Hsc70/Hsp70 and Hsp90. The chaperone activity of Hsc70/Hsp70 and Hsp90 occurs in coordinated cycles of ATP hydrolysis and substrate binding, and is regulated by a number of co-chaperone proteins. The import receptor Tom70 is a member of the tetratricopeptide repeat (TPR) co-chaperone family and contains a conserved TPR clamp domain for interaction with Hsc70 and Hsp90. Such interaction is essential for the initiation of the import process. This review will discuss the roles of Hsc70 and Hsp90 in mitochondrial import and summarize recent progress in understanding these pathways.

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.

Certain kinetoplastid (Leishmania spp. and Trypanosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.

Many Gram-negative bacteria are able to invade hosts by translocation of effectors directly into target cells in processes usually mediated by two very complex secretion systems (SSs), named type III (T3) and type IV (T4) SSs. These syringe-needle injection devices work with intervention of specialized secretion chaperones that, unlike traditional molecular chaperones, do not assist in protein folding and are not energized by ATP. Controversy still surrounds secretion chaperones primary role, but we can say that these chaperones act as: (i) bodyguards to prevent premature aggregation, or as (ii) pilots to direct substrate secretion through the correct secretion system. This family of chaperones does not share primary structure similarity but amazingly equal 3D folds. This mini review has the intent to present updated structural and functional data for several important secretion chaperones, either alone or in complex with their cognate substrates, as well to report on the common features and roles of T3, T4 and flagellar chaperones.

The present review intends to summarize the, yet preliminary, but very important emerging data underlining the functions exerted by the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1 on protein homeostasis. The main focus of the discussion is the cooperation between SIRT1 and the heat shock factor 1 (HSF1) responsible for activating the transcription of molecular chaperones, the protein-protective factors that resolve damaged/misfolded and aggregated proteins generated by heat stress or metabolism. SIRT1, a mammalian ortholog of the yeast silent information regulator 2, is a stress activated protein deacetylase that contributes to life-span extension by regulating different cell survival pathways, including replicative senescence, inflammation and resistance to hypoxic and heat stress. Phosphorylation is the major mechanism controlling the level and function of SIRT1 required for normal cell cycle progression and cell survival under stress conditions. Phosphorylated SIRT1 deacetylates and coactivates different substrates, including HSF1. Deacetylated HSF1 binds to the heat shock promoter element found upstream of genes encoding molecular chaperones. Overexpression of SIRT1 in cultured cells also helps them to survive exposure to heat stress. Conversely, its down-regulation accelerates the attenuation of the heat shock response promoting the release of HSF1 from its cognate promoter element. Very recently, in a mouse model for Alzheimer's disease, SIRT1 deacetylase activity was also found activating the transcription of α-secretase, the enzyme responsible for inhibiting the formation of aggregates of neuronal β-amyloid plaques. How SIRT1 activity protects cells from the deleterious effects of damaged/misfolded proteins and the implication of these findings on age-related pathologies are discussed.

One of the most important goals in bioinformatics is the ability to predict tertiary structure of a protein from its amino acid sequence. In this paper, new feature groups based on the physical and physicochemical properties of amino acids (size of the amino acids' side chains, predicted secondary structure based on normalized frequency of β-Strands, Turns, and Reverse Turns) are proposed to tackle this task. The proposed features are extracted using a modified feature extraction method adapted from Dubchak et al. To study the effectiveness of the proposed features and the modified feature extraction method, AdaBoost.M1, Multi Layer Perceptron (MLP), and Support Vector Machine (SVM) that have been commonly and successfully applied to the protein folding problem are employed. Our experimental results show that the new feature groups altogether with the modified feature extraction method are capable of enhancing the protein fold prediction accuracy better than the previous works found in the literature.

Protein palmitoylation is an important and common post-translational lipid modification of proteins and plays a critical role in various cellular processes. Identification of Palmitoylation sites is fundamental to decipher the mechanisms of these biological processes. However, experimental determination of palmitoylation residues without prior knowledge is laborious and costly. Thus computational approaches for prediction of palmitoylation sites in proteins have become highly desirable. Here, we propose PPWMs, a computational predictor using Position Weight Matrices (PWMs) encoding scheme and support vector machine (SVM) for identifying protein palmitoylation sites. Our PPWMs shows a nice predictive performance with the area under the ROC curve (AUC) of 0.9472 for the S-palmitoylation sites prediction and 0.9964 for the N-palmitoylation sites prediction on the newly proposed dataset. Comparison results show the superiority of PPWMs over two existing widely known palmitoylation site predictors CSS-Palm 2.0 and CKSAAP-Palm in many cases. Moreover, an attempt of incorporating structure information such as accessible surface area (ASA) and secondary structure (SS) into prediction is made and the structure characteristics are analyzed roughly. The corresponding software can be freely downloaded from http://math.cau.edu.cn/PPWMs.html.

A dimeric 64-kDa lectin was purified from seeds of French bean (Phaseolus vulgaris) cultivar number 1. The purification protocol entailed Q-Sepharose, Affi-gel blue gel, Mono S and Superdex 75. The lectin-enriched fraction was adsorbed on Q-Sepharose and Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Hemagglutinating activity was adsorbed on Mono S and eluted with a linear 0.3 - 1 M NaCl gradient. Gel filtration on Superdex 75 yielded a single absorbance peak which appeared as a single 32-kDa in sodium dodecyl sulfate poylacylamide gel electrophoresis. Full hemagglutinating activity was observed when the lectin was exposed to a pH ranging from 3 to 11. About 50% activity remained at pH 12, and about 25% at pH 0 to pH 2. Activity was totally abolished at pH 13 - 14. The activity was completely preserved when the ambient temperature was 20°C - 60°C. However, only 50% and 12.5% of the activity remained at 65°C and 70°C, respectively. Activity was barely discernible at 75°C and completely abrogated at and above 80°C. Hemagglutinating activity of the lectin was inhibited by glucuronic acid. Maximum mitogenic activity of the lectin toward murine splenocytes occurred at a lectin concentration of 0.488 μM. The mitogenic activity was nearly eliminated in the presence of 250 mM glucuronic acid. The lectin did not exhibit antiproliferative activity toward hepatoma (HepG2) cells, breast cancer (MCF7) cells, and nasopharynegeal carcinoma CNE stage 1 and stage 2 cells. It was also devoid of significant anti-HIV reverse transcriptase activity.

Water plays an invaluable role in governing the structure, stability, dynamics, and function of biomolecules, which has also been demonstrated to be critical in mediating biomolecular recognition and association. Accurate determination of the dynamic behavior of water molecules at biological complex interface is important for the understanding of the molecular mechanism of water contributing to the binding between biomolecules and could be exploited as an alternative tool to refine the water's positions in X-ray electron density map. In the present study, a method called local hydrophobic descriptors (LHDs) is used to characterize the hydrophobic landscapes of the hydration sites at protein-DNA interface. The resulting variables of the characterization are then correlated with the experimentally measured B-factor values of 4445 elaborately selected water samples derived from a panel of thematically nonredundant, high-quality protein-DNA interfaces by using a variety of machine learning methods, including PLS, BPNN, SVM, LSSVM, RF, and GP. The results show that the dynamic behavior of interfacial water molecules is primarily governed by the local hydrophobic feature of the hydration sites that water molecules located, and the nonlinear dependence dominates over the linear component in the water B-factor system. We expect that this structured-based approach can be used for predicting the B-factor profile of other biomolecules as well.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. Aims: To purify and characterize Thiol protease inhibitor from buffalo brain and to compare its properties with respect to tissue and organ difference from other mammalian cystatins. Main methods: Inhibitor has been isolated and purified using alkaline treatment; ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with a % yield of 64.13 and fold purification of 384.72. The inhibitor was studied by U.V and fluorescence spectroscopy .Papain inhibitory activity was measured using casein as substrate. Key finding: The molecular weight of the buffalo brain cystatin (BC), determined by gel filtration and SDS PAGE came out to be 43.6 KDa and 44.20 KDa respectively. BC was found to be stable in broad pH and temperature range. The inhibitor was devoid of any sulphydryl group and carbohydrate content. These properties led to conclusion that BC is variant of type-I cystatin. The stokes radius and diffusion coefficient of the inhibitor were found to be 27 A° and 8.1 10-7 cm /sec respectively, the f/f0 ratio was 1.12 signifying that purified cystatin is nearly globular in shape. Kinetic data revealed binding stoichiometry of BC with papain as 1:1. The Ki value with papain ficin and bromelain were found to be 1, 1.85 and 2.25nM respectively suggesting that cystatin has higher affinity with papain as compared to ficin and bromelain. The fluorescence and UV spectra of BC- papain complex showed significant conformational changes indicative of perturbation in the micro environment of aromatic amino acid residues on the formation of complex. Significance: This work proliferates our knowledge about cystatins of the mammalian brain on the basis of their physiochemical properties.