Protein and Peptide Letters - Volume 16, Issue 6, 2009

Machines in disrepair can either be fixed by mechanics or if they are completely damaged, they end up in a dumpster site. Proteins are machinery for function of an organism and must be strictly controlled for proper function. Heat shock proteins (Hsps) are the mechanics of almost all living organisms. By the same analogy, Hsps either help misfolded substrate proteins to reach their native conformation or send them for degradation [1]. Hsps function in a wide range of cellular processes; however their ultimate funct ion is to keep the proteins in the native conformation [1, 2]. Unfolded or partially folded proteins must reach a global free energy minimum to fold to their native state. The crowded molecular environment in a cell and hydrophobic environment of newly synthesized protein may cause protein aggregation, undesired interactions, and failure of assembling multi protein complexes [3]. Both prokaryotic and eukaryotic cells adapted a strategy to solve these problems; expressing heat shock proteins or so called “cellular mechanics”. Proposed models suggest that Hsp70s, Hsp40s and Hsp104 dissolve protein aggregates by acting together; therefore, emphasis was given on these proteins [3-5]. Hsp70 and Hsp104 were commonly studied in yeast, therefore Hsp70 and Hsp104 were further reviewed in S. cerevesiae by Dr. Jones and Dr. Chernoff, respectively. Several other Hsps are involved during substrate protein folding and prevention of aggregation. Because of their excessive number, these other proteins were not reviewed under separate topics but included under current topics. Hsp70 proteins interact with different Hsp40s to form a specialized function. Therefore, Hsp70 serves at a variety of cellular function. For this purpose, two leading groups working towards Hsp70 were invited to write reviews by emphasizing different aspects. The review by Dr. Jones specially focuses on yeast prions. The other review by Dr. Masison highlights structure-function relationship of Hsp70, Hsp70 interaction with other proteins, and Hsp70 role in signal transduction and apoptosis. Intensive research has been done on dissolving mechanism of the aggregates by heat shock proteins and Hsp70 is at the heart of this network. Hsp100 family chops off aggregates to facilitate Hsp70-Hsp40 complex function. Hsp104 chaperone role in interactions with aggregates and with Hsp70-Hsp40 complex in yeast was reviewed by Dr. Chernoff. Unique structure of Hsp100 serves to separate chunks from aggregates. Further biological functions of Hsp104, and structure of Hsp104 were reviewed by Dr. Glover. Hsp40 picks an unfolded protein and submits it to Hsp70. Hsp70 processes the unfolded substrate by providing a hydrophobic space. This allows a protein to fold to its native structure and makes an nonfunctional protein functional. Dr. Sha's group presented Hsp40 and its interaction with Hsp70. Classification and structure of Hsp40s were discussed in detail in order to better understand the folding of substrate peptides and solubilisation of aggregates. Throughout this mechanism new proteins were discovered, small Hsps and nucleotide exchange factors. Dr. Kocabiyik discusses small Hsps and their essential role for inhibiting aggregate formation. In the next review, Dr. Kabani provides an overview on nucleotide exchange factors. Hsp70 encapsulates and releases substrate proteins by coupling ATP hydrolysis energy. However to start the second round, hydrolyzed ATP must be removed from Hsp70 so that another ATP can bind. Nucleotide exchange factors replace ATP for ADP in Hsp70. Detailed information on nucleotide exchange factors are given in the review.

Heat shock proteins protect cells from various conditions of stress. Hsp70, the most ubiquitous and highly conserved Hsp, helps proteins adopt native conformation or regain function after misfolding. Various co-chaperones specify Hsp70 function and broaden its substrate range. We discuss Hsp70 structure and function, regulation by co-factors and influence on propagation of yeast prions.

Chaperones have long been recognised for their essential roles in the cell. They are involved in the refolding or degradation of misfolded proteins as well as the correct folding of newly synthesised proteins. However recent experiments have discovered that chaperones also have an important role to play in the propagation and maintenance of prions in yeast. The following minireview focuses on the Hsp70 chaperone family and it's involvement in the propagation of yeast prions.

Hsp104 is molecular chaperone in the AAA+ family of ATPases that specializes in the resolubilization and refolding of thermally denatured proteins in yeast. In addition to providing high levels of thermotolerance, Hsp104 plays a pivotal role in the propagation of yeast prions, self-replicating, amyloid-like aggregates that are inherited during mitosis and meiosis. In this review, the structure and function of Hsp104 is discussed, its functional interaction with other molecular chaperones, and a model for disaggregation and refolding is proposed.

High-ordered aggregates (amyloids) may disrupt cell functions, cause toxicity at certain conditions and provide a basis for self-perpetuated, protein-based infectious heritable agents (prions). Heat shock proteins acting as molecular chaperones counteract protein aggregation and influence amyloid propagation. The yeast Hsp104/Hsp70/Hsp40 chaperone complex plays a crucial role in interactions with both ordered and unordered aggregates. The main focus of this review will be on the Hsp104 chaperone, a molecular “disaggregase”.

The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question, as is how Hsp40 co-operates with Hsp70 to facilitate protein folding. Years of structural studies of Hsp40 from yeast and other species, conducted using X-ray protein crystallography, NMR and small-angle X-ray scattering, have shed light on the mechanisms how Hsp40 functions as a molecular chaperone and how Hsp40- Hsp70 pair promotes protein folding, protein transport and degradation. This review provides a discussion of recent structural studies of Hsp40s and their functional implications.

Small heat shock proteins are ubiquitously found in all three domains of life, although they are the most poorly conserved family of molecular chaperones. Their involvement in anti-stress mechanisms of the cells have been clearly demonstrated by induction of their expression in response to various environmental and pathological stresses. Small heat shock proteins comprise the most effective chaperone family concerning their unusual capacity of substrate binding. It is well documented that small heat shock proteins associate with unfolding substrate proteins and form large oligomeric complexes to prevent their aggregation and accumulation, that otherwise would impair the normal cell functions. The substrates captured by small heat shock proteins are further refolded to their native state by ATP depended chaperones. During heat stress, the induced expression and activation of the small heat shock proteins, might reflect that this mechanism of protein quality control contributes to acquired thermotolerance in hyperthermophilic archaea, as well.

Since their recent identification, eukaryotic Hsp70 nucleotide exchange factors (NEFs) have gained increasing interest due to their engagement in vital cellular processes. Here, I summarize our current knowledge of their mechanisms of action, regulations and cellular functions as well as their relevance for human diseases such as cystic fibrosis or amyloidoses.

This article focuses on ribosome-associated chaperones. A chaperone bound close to the exit tunnel on a ribosome 25 Å from the emerging nascent chain has an effective concentration of 1 x 10^-1 M, which is 4-5 orders of magnitude larger than the concentration of the chaperone in the cytosol. Ribosome-bound chaperones bind nascent chains intramolecularly with rates as large as 10⁴ s^-1 in order to keep chains unfolded.

Fluorescent live cell imaging has recently been used in numerous studies to examine prions in yeast. These fluorescence studies take advantage of the fact that unlike the normally folded form, the misfolded amyloid form of the prion protein is aggregated. The studies have used fluorescence to identify new prions, to study the transmission of prion from mother to daughter, and to understand the role of molecular chaperones in this transmission. The use of fluorescence imaging complements the more standard methods used to study prion propagation. This review discusses the various studies that have taken advantage of fluorescence imaging technique particularly in regard to understanding the transmission and curing of the [PSI⁺], the prion form of the translation termination factor Sup35p.

The Hsp90 and Hsp70 molecular chaperones play important roles in the folding and proper functioning of diverse cellular proteins, including transcriptional regulators and protein kinases. In yeast, several transcriptional regulators and protein kinases are known to be substrates for Hsp90 and Hsp70 molecular chaperones. The yeast heme activator protein Hap1 promotes transcription of many genes in response to heme. It requires Hsp90 and Hsp70 molecular chaperones for its activity to be precisely regulated by heme concentration. The mechanism by which molecular chaperones promote heme regulation of Hap1 activity is distinct from the mechanism by which molecular chaperones promote steroid signaling. Hsp70 and Hsp90 molecular chaperones act separately to promote Hap1 repression in heme-deficient cells and heme activation of Hap1 in heme-sufficient cells. Likewise, distinct Hap1 elements or domains act to mediate Hap1 repression and heme activation separately. In this review, we summarize the current knowledge about the molecular mechanism governing heme regulation of Hap1 activity, and we compare this mechanism to the molecular mechanism by which Hsp90 and Hsp70 molecular chaperones promote the regulation of glucocorticoid receptor, the most extensively studied substrate of Hsp90 and Hsp70 molecular chaperones.

The gastrin-releasing peptide receptor (GRPR) is a therapeutic target in colon cancer. Here we show that the GRPR antagonist RC-3095 (10^-3, 10^-6, or 1 μM) decreases nerve growth factor (NGF) secretion measured by enzymelinked immunosorbent assay (ELISA) in HT-29 human colon carcinoma cells. The results suggest that decreased secretion of neurotrophins might be a novel mechanism by which GRPR antagonists exert their antiproliferative effects in cancer cells.

The effects of β-cyclodextrin (β-CyD) and trehalose on glycation of human serum albumin (HSA) were studied. These additives reduced AGEs and nanofibril formation of HSA under in vitro glycation conditions and improved its helical structure. These were accomplished through direct interactions of them with HSA and alterations in solute-protein interactions.

C-terminal fragment of the Botulinum neurotoxin A comprises two sub-domains including HC-N and HC-C. Here, the conformational change of HC-N was studied by spectroscopic techniques. The results indicated that the partially unfolded state forms during unfolding of HC-N. This finding may shed light on poorly - known features of the protein.

The engineering of a novel FGF2 fused with highly conserved bone mineral-binding domain of osteocalcin (OC) for targeting to bone mineral hydroxyapatite (HA) exhibited much stronger HA-binding affinity than native FGF2. FGF2-OC also showed a significant increase of mitogenic activity and cellular differentiation of osteoblastic cells compared with native FGF2.

Human β-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 β-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed ¹H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.

The glycan-binding profile of a β-galactoside-binding 15 kDa lectin (Galectin-1) purified from the oocytes of the American bullfrog, Rana catesbeiana, was studied using 61 pyridyl-aminated oligosaccharides by frontal affinity chromatography. Human blood type-A-hexasaccharide (GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-4Galβ1-4Glc) was found to exhibit the strongest ligand binding to the galectin while Forssman antigen (GalNAcα1-3GalNAcβ1-3Galα1- 4Galβ1-4Glc) and type-A-tetrasaccharide (GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-4Glc) were also extensively recognized. The kinetics of affinity of galectin-1 to type-A oligosaccharide was analysed by surface plasmon resonance using neoglycoprotein with type-A oligosaccharides. R. catesbeiana oocyte galectin adhered to human rhabdomyosarcoma cells dose dependently and the activity was specifically cancelled by the neoglycoprotein. It was concluded that galectin-1 from R. catesbeiana oocytes possesses different and rare glycan-binding properties from typical members in galectin family.

To investigate the function of plant plasma membrane, proteins of rice plasma membrane were analyzed and the proteins changed by cold stress were identified. Plasma membrane proteins were purified with an aqueous two-phase partitioning method from root of rice seedlings, and activity of specific H⁺-ATPase localized in plasma membranes was measured. The plasma membrane proteins were separated by SDS-PAGE or 2D-PAGE, and analyzed with nano LCMS/ MS. The number of transmembrane helices was predicted from the amino acid sequence of annotated proteins. Functional categorization revealed that the most of proteins were associated with energy production, signal transduction, protein synthesis, cell growth/division and defense. In addition, 12 cold stress responsive proteins were identified from the plasma membrane using 2D-PAGE based proteomics method. Out of them, cold shock protein-1 was significantly decreased in plasma membrane of rice under cold stress.

To further clarify the priming mechanism of liver regeneration, proteins and protein complexes from rat plasma (normal group, partial hepatectomy (PHx) group and sham-operation group) were comparatively studied by twodimensional gel electrophoresis and two-dimensional blue native gel electrophoresis. Our results suggested that Kupffer cell—NF-κB/ROS might trigger the liver regeneration.

The twin-arginine translocation (Tat) pathway is an attractive route for secretory production of heterologous proteins in E. coli. In this study, we investigated the potential use of Tat signal peptide from S. coelicolor to improve secretory expression. The results showed that Tat signal peptide (ssDagA) could effectively secrete active Green fluorescent protein (GFP) to periplasm. When the rare codons of signal sequence were optimized, the expression and secretion yield of GFP improved by about 2-3 folds as detected qualitatively by western blotting and fluorescent analysis. The increase of translation rate could be explained by the unstability of mRNA secondary structure. In summary, our strategy could provide a new approach for high-level secretory expression of heterologous proteins in E. coli.