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European Journal of Clinical... May 2021In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches... (Review)
Review
BACKGROUND
In the last decades, cardiovascular diseases (CVD) have remained the first leading cause of mortality and morbidity in the world. Although several therapeutic approaches have been introduced in the past, the development of novel treatments remains an important research goal, which is hampered by the lack of understanding of key mechanisms and targets. Emerging evidences in recent years indicate the involvement of misfolded proteins aggregation and the derailment of protein quality control in the pathogenesis of cardiovascular diseases. Several potential interventions targeting protein quality control have been translated from the bench to the bedside to effectively employ the misfolded proteins as promising therapeutic targets for cardiac diseases, but with trivial results.
DESIGN
In this review, we describe the recent progresses in preclinical and clinical studies of protein misfolding and compromised protein quality control by selecting and reporting studies focusing on cardiovascular diseases including cardiomyopathies, cardiac amyloidosis, atherosclerosis, atrial fibrillation and thrombosis.
RESULTS
In preclinical models, modulators of several molecular targets (eg heat shock proteins, unfolded protein response, ubiquitin protein system, autophagy and histone deacetylases) have been tested in various conditions with promising results although lacking an adequate transition towards clinical setting.
CONCLUSIONS
At present, no therapeutic strategies have been reported to attenuate proteotoxicity in patients with CVD due to a lack of specific biomarkers for pinpointing upstream events in protein folding defects at a subclinical stage of the diseases requiring an intensive collaboration between basic scientists and clinicians.
Topics: Amyloidosis; Animals; Atherosclerosis; Atrial Fibrillation; Autophagy; Cardiomyopathies; Cardiovascular Diseases; Heat-Shock Proteins; Histone Deacetylases; Humans; Protein Aggregation, Pathological; Protein Folding; Protein Refolding; Proteostasis; Proteostasis Deficiencies; Thrombosis; Ubiquitination; Unfolded Protein Response
PubMed: 33527342
DOI: 10.1111/eci.13504 -
Methods in Molecular Biology (Clifton,... 2023Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The...
Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The reason for this phenomenon is found in the reducing environment of the cytoplasm, preventing the formation of disulfide bridges and therefore resulting in inactive protein aggregates. However, IBs can be refolded in vitro to obtain the protein in its active conformation. In order to correctly form the required disulfide bridges, cystines are fully reduced during solubilization and, with the help of an oxidizing agent, the native disulfide bridges are formed during the refolding step. Here, a protocol to identify suitable redox conditions for solubilization and refolding is presented. For this purpose, a multivariate approach spanning the unit operations solubilization and refolding is used.
Topics: Disulfides; Escherichia coli; Oxidation-Reduction; Protein Folding; Protein Refolding; Recombinant Proteins; Solubility; Inclusion Bodies
PubMed: 36656523
DOI: 10.1007/978-1-0716-2930-7_11 -
Frontiers in Bioengineering and... 2023Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant... (Review)
Review
Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.
PubMed: 37545893
DOI: 10.3389/fbioe.2023.1249196 -
Biophysical Reviews Aug 2023Metamorphic proteins are a paradigm of the protein folding process, by encoding two or more native states, highly dissimilar in terms of their secondary, tertiary, and... (Review)
Review
Metamorphic proteins are a paradigm of the protein folding process, by encoding two or more native states, highly dissimilar in terms of their secondary, tertiary, and even quaternary structure, on a single amino acid sequence. Moreover, these proteins structurally interconvert between these native states in a reversible manner at biologically relevant timescales as a result of different environmental cues. The large-scale rearrangements experienced by these proteins, and their sometimes high mass interacting partners that trigger their metamorphosis, makes the computational and experimental study of their structural interconversion challenging. Here, we present our efforts in studying the refolding landscapes of two quintessential metamorphic proteins, RfaH and KaiB, using simplified dual-basin structure-based models (SBMs), rigorously footed on the energy landscape theory of protein folding and the principle of minimal frustration. By using coarse-grained models in which the native contacts and bonded interactions extracted from the available experimental structures of the two native states of RfaH and KaiB are merged into a single Hamiltonian, dual-basin SBM models can be generated and savvily calibrated to explore their fold-switch in a reversible manner in molecular dynamics simulations. We also describe how some of the insights offered by these simulations have driven the design of experiments and the validation of the conformational ensembles and refolding routes observed using this simple and computationally efficient models.
PubMed: 37681096
DOI: 10.1007/s12551-023-01087-0 -
Annual Review of Microbiology Sep 2019Small heat shock proteins (sHsps) constitute a diverse chaperone family that shares the α-crystallin domain, which is flanked by variable, disordered N- and C-terminal... (Review)
Review
Small heat shock proteins (sHsps) constitute a diverse chaperone family that shares the α-crystallin domain, which is flanked by variable, disordered N- and C-terminal extensions. sHsps act as the first line of cellular defense against protein unfolding stress. They form dynamic, large oligomers that represent inactive storage forms. Stress conditions cause a rapid increase in cellular sHsp levels and trigger conformational rearrangements, resulting in exposure of substrate-binding sites and sHsp activation. sHsps bind to early-unfolding intermediates of misfolding proteins in an ATP-independent manner and sequester them in sHsp/substrate complexes. Sequestration protects substrates from further uncontrolled aggregation and facilitates their refolding by ATP-dependent Hsp70-Hsp100 disaggregases. Some sHsps with particularly strong sequestrase activity, such as yeast Hsp42, are critical factors for forming large, microscopically visible deposition sites of misfolded proteins in vivo. These sites are organizing centers for triaging substrates to distinct quality control pathways, preferentially Hsp70-dependent refolding and selective autophagy.
Topics: Adenosine Triphosphate; Heat-Shock Proteins, Small; Hot Temperature; Protein Folding; Protein Multimerization; Stress, Physiological
PubMed: 31091419
DOI: 10.1146/annurev-micro-020518-115515 -
International Journal of Molecular... Sep 2019Heat shock proteins (HSPs) are associated with various physiological processes (protein refolding and degradation) involved in the responses to cellular stress, such as... (Review)
Review
Heat shock proteins (HSPs) are associated with various physiological processes (protein refolding and degradation) involved in the responses to cellular stress, such as cytotoxic agents, high temperature, and hypoxia. HSPs are overexpressed in cancer cells and play roles in their apoptosis, invasion, proliferation, angiogenesis, and metastasis. The regulation or translational modification of HSPs is recognized as a therapeutic target for the development of anticancer drugs. Among the regulatory processes associated with HSP expression, the epigenetic machinery (miRNAs, histone modification, and DNA methylation) has key functions in cancer. Moreover, various epigenetic modifiers of HSP expression have also been reported as therapeutic targets and diagnostic markers of cancer. Thus, in this review, we describe the epigenetic alterations of HSP expression in cancer cells and suggest that HSPs be clinically applied as diagnostic and therapeutic markers in cancer therapy via controlled epigenetic modifiers.
Topics: Animals; Apoptosis; Biomarkers, Tumor; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Histones; Humans; Methylation; MicroRNAs; Molecular Targeted Therapy; Neoplasms; Protein Processing, Post-Translational
PubMed: 31557887
DOI: 10.3390/ijms20194758 -
Cell Death & Disease Sep 2020Carboxy-terminus of Hsc70-interacting protein (CHIP) functions both as a molecular co-chaperone and ubiquitin E3 ligase playing a critical role in modulating the... (Review)
Review
Carboxy-terminus of Hsc70-interacting protein (CHIP) functions both as a molecular co-chaperone and ubiquitin E3 ligase playing a critical role in modulating the degradation of numerous chaperone-bound proteins. To date, it has been implicated in the regulation of numerous biological functions, including misfolded-protein refolding, autophagy, immunity, and necroptosis. Moreover, the ubiquitous expression of CHIP in the central nervous system suggests that it may be implicated in a wide range of functions in neurological diseases. Several recent studies of our laboratory and other groups have highlighted the beneficial role of CHIP in the pathogenesis of several neurological diseases. The objective of this review is to discuss the possible molecular mechanisms that contribute to the pathogenesis of neurological diseases in which CHIP has a pivotal role, such as stroke, intracerebral hemorrhage, Alzheimer's disease, Parkinson's disease, and polyglutamine diseases; furthermore, CHIP mutations could also cause neurodegenerative diseases. Based on the available literature, CHIP overexpression could serve as a promising therapeutic target for several neurological diseases.
Topics: Animals; Humans; Nervous System Diseases; Neurodegenerative Diseases; Ubiquitin-Protein Ligases
PubMed: 32908122
DOI: 10.1038/s41419-020-02953-5 -
International Journal of Biological... Jan 2021The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed...
The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed for various applications, such as drug delivery, molecular imaging, and liquid biopsy etc. However, the challenges remain to ensure protein stability and to retain the conformational state of the protein intact upon nano-conjugation. In this communication we have reported the status of stability and refolding ability of Au-NP conjugated zDHFR protein. The effect of nano-conjugation of zDHFR on the thermal stability and it's refolding from thermally denatured state have been extensively studied. Zebrafish Dihydrofolate reductase (zDHFR) is an essential enzyme which acts as a crucial part in synthesis of purine, thymidylate and various amino acids in cells. We have nano-conjugated zDHFR protein with Au-nanoparticles and studies were conducted for thermally denatured Au-NP conjugated zDHFR and compared with the non-conjugated protein. Refolding experiment of heat denatured Au-NP conjugated zDHFR was carried out to check the status of refolding and the result was compared with the non-conjugated protein. Our observation reveals that nano-conjugation stabilises the zDHFR protein against thermal denaturation. Furthermore, the nano-conjugation promotes refolding process of thermally unfolded DHFR such that the yield of refolding substantially increases.
Topics: Animals; Chemical Phenomena; Gene Expression; Gold; Kinetics; Metal Nanoparticles; Nanostructures; Protein Denaturation; Protein Folding; Protein Refolding; Protein Stability; Recombinant Proteins; Tetrahydrofolate Dehydrogenase; Thermodynamics; Zebrafish
PubMed: 33181215
DOI: 10.1016/j.ijbiomac.2020.11.053 -
Antibody Therapeutics Oct 2023Arginine (Arg) is a natural amino acid with an acceptable safety profile and a unique chemical structure. Arg and its salts are highly effective in enhancing protein... (Review)
Review
Arginine (Arg) is a natural amino acid with an acceptable safety profile and a unique chemical structure. Arg and its salts are highly effective in enhancing protein refolding and solubilization, suppressing protein-protein interaction and aggregation and reducing viscosity of high concentration protein formulations. Arg and its salts have been used in research and 20 approved protein injectables. This review summarizes the effects of Arg as an excipient in therapeutic protein formulations with the focus on its physicochemical properties, safety, applications in approved protein products, beneficial and detrimental effects in liquid and lyophilized protein formulations when combined with different counterions and mechanism on protein stabilization and destabilization. The decade literature review indicates that the benefits of Arg overweigh its risks when it is used appropriately. It is recommended to add Arg along with glutamate as a counterion to high concentration protein formulations on top of sugars or polyols to counterbalance the negative effects of Arg hydrochloride. The use of Arg as a viscosity reducer and protein stabilizer in high concentration formulations will be the inevitable future trend of the biopharmaceutical industry for subcutaneous administration.
PubMed: 38075239
DOI: 10.1093/abt/tbad022 -
Frontiers in Molecular Biosciences 2021Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress.... (Review)
Review
Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress. sHsps are defined not only by their relatively low molecular weight, but also by the presence of a conserved α-crystallin domain, which is flanked by less conserved, mostly unstructured, N- and C-terminal domains. sHsps form oligomers of different sizes which deoligomerize upon stress conditions into smaller active forms. Activated sHsps bind to aggregation-prone protein substrates to form assemblies that keep substrates from irreversible aggregation. Formation of these assemblies facilitates subsequent Hsp70 and Hsp100 chaperone-dependent disaggregation and substrate refolding into native species. This mini review discusses what is known about the role and place of bacterial sHsps in the chaperone network.
PubMed: 34055885
DOI: 10.3389/fmolb.2021.666893