Highly Precise Protein Semisynthesis through Ligation-Desulfurization Chemistry in Combination with Phenacyl Protection of Native Cysteines.

Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. The development of various ß-mercapto amino acid building blocks coupled with ligation-desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation-desulfurization sequence.

Advances in the Development of Anticancer HSP-based Vaccines.

Current advances in cancer treatment are based on the recent discoveries of molecular mechanisms of tumour maintenance. It was shown that heat shock proteins (HSPs) play a crucial role in the development of immune response against tumours. Thus, HSPs represent multifunctional agents not only with chaperone functions, but also possessing immunomodulatory properties. These properties are exploited for the development of HSP-based anticancer vaccines aimed to induce cytotoxic responses against tumours. To date, a number of strategies have been suggested to facilitate HSP-based vaccine production and to increase its effectiveness. The present review focuses on the current trend for the development of HSPbased vaccines aimed at inducing strong immunological tumour-specific responses against cancer cells of distinct etiology and localization.

A fragment of the Escherichia coli ClpB heat-shock protein is a micromolar melanocortin 1 receptor agonist.

The melanocortin system consists of five receptor subtypes (MC1-5R), endogenous agonists derived from the proopiomelanocortin gene transcript, and the antagonists agouti and agouti-related protein. The Escherichia coli heat shock protein ClpB has previously been described as an antigen mimetic to the endogenous melanocortin agonist α-MSH. Herein, we investigated if a fragment of the ClpB protein could directly signal through the melanocortin receptors. We synthesized a complementary fragment of the ClpB protein that partially aligned with α-MSH. Pharmacological assessment of this fragment resulted in no antagonist activity at the MC3R or the MC4R and no agonist activity at the MC4R. Partial receptor activation was observed for the MC3R and MC5R at 100 µM concentrations. This fragment was shown to be a full micromolar MC1R agonist and may serve as a template for future research into selective MC1R ligands.

Repurposing Hsp104 to Antagonize Seminal Amyloid and Counter HIV Infection.

Naturally occurring proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) form amyloid fibrils in seminal fluid, which capture HIV virions and promote infection. For example, PAP248-286 fibrils, termed SEVI (semen-derived enhancer of viral infection), can potentiate HIV infection by several orders of magnitude. Here, we design three disruptive technologies to rapidly antagonize seminal amyloid by repurposing Hsp104, an amyloid-remodeling nanomachine from yeast. First, Hsp104 and an enhanced engineered variant, Hsp104(A503V), directly remodel SEVI and PAP85-120 fibrils into non-amyloid forms. Second, we elucidate catalytically inactive Hsp104 scaffolds that do not remodel amyloid structure, but cluster SEVI, PAP85-120, and SEM1(45-107) fibrils into larger assemblies. Third, we modify Hsp104 to interact with the chambered protease ClpP, which enables coupled remodeling and degradation to irreversibly clear SEVI and PAP85-120 fibrils. Each strategy diminished the ability of seminal amyloid to promote HIV infection, and could have therapeutic utility.

Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate.

One of the most important requirements for non-viral gene delivery systems is the ability to mediate high levels of gene expression with low toxicity. After the DNA/vector complexes are taken up by cells through endocytosis, DNA is typically contained within the endocytic compartments and rapidly degraded due to the low pH and hydrolytic enzymes within endosomes and lysosomes, limiting its accessibility to the cytosol and ultimately to the nucleus. In this study, the endosomolytic protein listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes was conjugated with polyethylenimine (PEI) of average molecular weight 25 kDa (PEI25) via a reversible disulfide bond (LLO-s-s-PEI), and incorporated into plasmid DNA condensed with disulfide-crosslinked low molecular weight PEI 1.8 kDa (PEI1.8). We have investigated and demonstrated that high gene transfection efficiency, which is comparable to that by the most commonly used PEI25, can be achieved by reversibly crosslinking low molecular weight PEI (PEI1.8) using disulfide bonds, with greatly reduced cytotoxicity of the PEI. The reversible incorporation of LLO into the DNA condensates of PEI, through the use of the synthesized LLO-s-s-PEI conjugate, further enhances the transfection efficiency beyond that of DNA condensates with disulfide-crosslinked PEI1.8 alone.

Thermoresponsive controlled association of protein with a dynamic nanogel of hydrophobized polysaccharide and cyclodextrin: heat shock protein-like activity of artificial molecular chaperone.

Dynamic CHP-CD nanogels, which consisted of a self-assembly of cholesteryl-group-bearing pullulan (CHP) and beta-cyclodextrin (CD), were characterized by SEC and SEC-MALS methods. The nanogels prevented the thermal aggregation of carbonic anhydrase B (CAB) by selective trapping of the heat-denatured protein. After the complex between the CHP-CD nanogels and CAB was cooled, the enzyme activity of CAB spontaneously recovered upon release from the complex. The dynamic nanogels self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD. The thermal stability of CAB was improved by thermoresponsive controlled association between the proteins and the artificial molecular chaperone.

Efeito da temperatura na síntese de proteínas em células de adenocarcinoma de pulmäo (A549) pré-tratadas com interferon / Effect of temperature on proteins synthesis in human lung adenocarcinoma cells (A549) pre-treated with interferon

No presente trabalho foi investigado o efeito da temperatura elevada na síntese de proteínas em células de adenocarcinoma de pulmao (A549) previamente tratadas com Interferon recombinante humano alpha-2b. Os resultados mostram que em nossas condiçoes experimentais, o pré-tratamento das células com esta droga inibe a expressao dos genes de choque térmico.

A synthetic peptide administered with IL-12 elicits immunity to Listeria monocytogenes.

IL-12 is a pivotal cytokine signal for the development of Th1-type cellular responses that are required for control of intracellular pathogens. We previously demonstrated that coinjection of IL-12 with heat-killed Listeria monocytogenes, which was not immunogenic when injected alone, elicited intense Ag-specific T cell responses that conferred protection against subsequent challenge with Listeria. Herein we describe the remarkable finding that a nonimmunogenic synthetic peptide corresponding to a dominant MHC class II (H-2k)-restricted listerial determinant, when coinjected i.p. with murine IL-12, elicited potent Ag-specific immune responses that conferred protective immunity against Listeria.

Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.

Chemical synthesis of 10 kDa chaperonin. Biological activity suggests chaperonins do not require other molecular chaperones.

Molecular chaperones are required for the correct folding and assembly of certain other polypeptides. It is not known whether molecular chaperones themselves require other chaperones to become functional. A 97-amino acid chaperone, the chaperonin 10 protein was chemically synthesised so that during synthesis and purification there was no contact of the chaperone with any other protein. The purified, synthetic chaperonin 10 protein formed oligomeric structures spontaneously and was biologically active as a chaperonin. This is the first description of a chemically synthesised chaperonin, and suggests that no other chaperones are required for correct folding, polymerisation and biological activity of this chaperone.

Modulation for antigen presentation in tuberculosis by using synthetic peptides.

Competition assay technology has been a very useful tool in the study of parasite antigens and has been inferred but never proven that this approach can be applied to select T-cell epitopes by using another microorganisms. In this study, HLA-restricted T-cell clones specific to synthetic peptides derived from the 65 kDa mycobacterial protein were used to investigate whether these peptides are able to compete with each other at the level of MHC-binding sites in tuberculosis. Fixed APCs were pulsed with suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptide. The results showed that two peptides from this protein were able to compete with each other inducing a significant inhibition in the proliferation assays while there was no competition by using a control peptide. The amount of cross-reactivity was influenced by the peptide concentrations. More important was the observation that these peptides were able to bind to the same HLA-class II molecules therefore blocking the binding of each other. The fact that these peptides have not an identical amino acid sequence support the idea that the MHC-peptide interaction must have a broad specificity to be able to bind a large number of peptides. These results demonstrate that it is possible to modulate the antigen presentation by blocking the peptide MHC-class II interaction in tuberculosis and support the idea that this approach facilitates the selection of appropriate T-cell epitopes to be incorporated in a vaccine.

Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein.

Adjuvant arthritis in Lewis rats is a model of T cell-mediated autoimmune arthritis resembling human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium bovis BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-terminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cell-specific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.

Trimerization of a yeast transcriptional activator via a coiled-coil motif.

The transcriptional induction of heat shock genes is mediated by the heat shock transcription factor (HSF). We have investigated the oligomerization state of HSF from S. cerevisiae and find that it forms a trimer in solution and when bound to DNA. Trimerization is mediated by a region of HSF that, like the leucine zipper, is characterized by the occurrence of hydrophobic amino acids every 7 residues. We propose that it forms a three-stranded coiled coil.

In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.

In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.