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International Journal of General... 2024High temperature requirement A1 (HTRA1) is a member of the serine protease family, comprising four structural domains: IGFBP domain, Kazal domain, protease domain and... (Review)
Review
High temperature requirement A1 (HTRA1) is a member of the serine protease family, comprising four structural domains: IGFBP domain, Kazal domain, protease domain and PDZ domain. HTRA1 encodes a serine protease, a secreted protein that is widely expressed in the vasculature. HTRA1 regulates a wide range of physiological processes through its proteolytic activity, and is also involved in a variety of vascular abnormalities-related diseases. This article reviews the role of HTRA1 in the development of vascular abnormalities-related hereditary cerebral small vessel disease (CSVD), age-related macular degeneration (AMD), tumors and other diseases. Through relevant research advances to understand the role of HTRA1 in regulating signaling pathways or refolding, translocation, degradation of extracellular matrix (ECM) proteins, thus directly or indirectly regulating angiogenesis, vascular remodeling, and playing an important role in vascular homeostasis, further understanding the mechanism of HTRA1's role in vascular abnormality-related diseases is important for HTRA1 to be used as a therapeutic target in related diseases.
PubMed: 38650587
DOI: 10.2147/IJGM.S456912 -
Nature Communications Apr 2024Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or...
Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.
Topics: DNA Damage; Heat-Shock Proteins, Small; Intranuclear Inclusion Bodies; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Vesicular Transport Proteins
PubMed: 38615096
DOI: 10.1038/s41467-024-47615-8 -
International Journal of Molecular... Apr 2024Refolding multi-disulfide bonded proteins expressed in into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and...
Refolding multi-disulfide bonded proteins expressed in into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the N-HSQC NMR spectrum exhibited several H-N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.
Topics: Humans; Influenza, Human; Escherichia coli; Influenza A Virus, H1N1 Subtype; Orthomyxoviridae Infections; Disulfides
PubMed: 38612753
DOI: 10.3390/ijms25073943 -
Frontiers in Molecular Biosciences 2024COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the...
COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the world. The viral positive-sense single-stranded RNA encodes 31 proteins among which the spike (S) is undoubtedly the best known. Recently, protein E has been reputed as a potential pharmacological target as well. It is essential for the assembly and release of the virions in the cell. Literature describes protein E as a voltage-dependent channel with preference towards monovalent cations whose intracellular expression, though, alters Ca homeostasis and promotes the activation of the proinflammatory cascades. Due to the extremely high sequence identity of SARS-CoV-2 protein E (E-2) with the previously characterized E-1 (i.e., protein E from SARS-CoV) many data obtained for E-1 were simply adapted to the other. Recent solid state NMR structure revealed that the transmembrane domain (TMD) of E-2 self-assembles into a homo-pentamer, albeit the oligomeric status has not been validated with the full-length protein. Prompted by the lack of a common agreement on the proper structural and functional features of E-2, we investigated the specific mechanism/s of pore-gating and the detailed molecular structure of the most cryptic protein of SARS-CoV-2 by means of MD simulations of the E-2 structure and by expressing, refolding and analyzing the electrophysiological activity of the transmembrane moiety of the protein E-2, in its full length. Our results show a clear agreement between experimental and predictive studies and foresee a mechanism of activity based on Ca affinity.
PubMed: 38606285
DOI: 10.3389/fmolb.2024.1334819 -
Journal of Animal Science Jan 2024Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study...
Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study investigated the hypothesis that HS alters the ovarian proteome and negatively impacts proteins engaged with insulin signaling, inflammation, and ovarian function. Prepubertal gilts (n = 19) were assigned to one of three environmental groups: thermal neutral with ad libitum feed intake (TN; n = 6), thermal neutral pair-fed (PF; n = 6), or HS (n = 7). For 7 d, HS gilts were exposed to 12-h cyclic temperatures of 35.0 ± 0.2 °C and 32.2 ± 0.1 °C, while TN and PF gilts were housed at 21.0 ± 0.1 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on ovarian protein homogenates. Relative to TN gilts, 178 proteins were altered (P ≤ 0.05, log2foldchange ≥ 1) by HS, with 76 increased and 102 decreased. STRING gene ontology classified and identified 45 biological processes including those associated with chaperone protein refolding, cytoplasmic translational initiation, and immune activation; with a protein-protein interaction web network of 158 nodes and 563 edges connected based on protein function (FDR ≤ 0.05). Relative to PF, HS altered 330 proteins (P ≤ 0.05, log2foldchange ≥ 1), with 151 increased and 179 decreased. Fifty-seven biological pathways associated with protein function and assembly, RNA processing, and metabolic processes were identified, with a protein-protein interaction network of 303 nodes and 1,606 edges. Comparing HS with both the TN and PF treatments, 72 ovarian proteins were consistently altered by HS with 68 nodes and 104 edges, with biological pathways associated with translation and gene expression. This indicates that HS alters the ovarian proteome and multiple biological pathways and systems in prepubertal gilts; changes that potentially contribute to female infertility.
Topics: Swine; Female; Animals; Proteome; Chromatography, Liquid; Tandem Mass Spectrometry; Sus scrofa; Heat-Shock Response; Hot Temperature
PubMed: 38605681
DOI: 10.1093/jas/skae053 -
Analytical and Bioanalytical Chemistry May 2024Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of...
Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.
Topics: Inclusion Bodies; Protein Refolding; Spectrometry, Fluorescence; Recombinant Proteins; Tryptophan; Escherichia coli; Tyrosine; Fluorescence; Protein Folding
PubMed: 38573344
DOI: 10.1007/s00216-024-05249-1 -
Journal of Microbiology and... May 2024The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to... (Comparative Study)
Comparative Study
The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of : i) engineered strains with deletions in the disulfide reduction pathways, SHuffle, and ii) the co-expression of oxidative folding catalysts, CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.
Topics: Escherichia coli; Disulfides; Cytoplasm; Recombinant Proteins; Protein Folding; Escherichia coli Proteins; Oxidation-Reduction; Protein Disulfide-Isomerases; Periplasm; Culture Media
PubMed: 38563095
DOI: 10.4014/jmb.2311.11025 -
Vaccines Mar 2024Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in...
Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in , the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.
PubMed: 38543900
DOI: 10.3390/vaccines12030267 -
Pharmaceuticals (Basel, Switzerland) Feb 2024Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve...
Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.
PubMed: 38543088
DOI: 10.3390/ph17030302 -
Current Opinion in Structural Biology Jun 2024In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which... (Review)
Review
In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which reversibly interconvert between multiple, sometimes highly dissimilar, native states. As the number of known metamorphic proteins increases, several computational and experimental strategies have emerged for gaining insights about their refolding processes and identifying unknown metamorphic proteins amongst the known proteome. In this review, we describe the current advances in biophysically and functionally ascertaining the structural interconversions of metamorphic proteins and how coevolution can be harnessed to identify novel metamorphic proteins from sequence information. We also discuss the challenges and ongoing efforts in using artificial intelligence-based protein structure prediction methods to discover metamorphic proteins and predict their corresponding three-dimensional structures.
Topics: Proteins; Protein Folding; Protein Conformation; Models, Molecular; Humans; Artificial Intelligence
PubMed: 38537533
DOI: 10.1016/j.sbi.2024.102807