-
Cell Stress & Chaperones Jun 2024More than 99% of the mitochondrial proteome is encoded by the nucleus and requires refolding following import. Therefore, mitochondrial proteins require the coordinated... (Review)
Review
More than 99% of the mitochondrial proteome is encoded by the nucleus and requires refolding following import. Therefore, mitochondrial proteins require the coordinated action of molecular chaperones for their folding and activation. Several heat shock protein (Hsp) molecular chaperones, including members of the Hsp27, Hsp40/70, and Hsp90 families, as well as the chaperonin complex Hsp60/10 have an established role in mitochondrial protein import and folding. The "Chaperone Code" describes the regulation of chaperone activity by dynamic post-translational modifications; however, little is known about the post-translational regulation of mitochondrial chaperones. Dissecting the regulation of chaperone function is essential for understanding their differential regulation in pathogenic conditions and the potential development of efficacious therapeutic strategies. Here, we summarize the recent literature on post-translational regulation of mitochondrial chaperones, the consequences for mitochondrial function, and potential implications for disease.
Topics: Humans; Mitochondria; Molecular Chaperones; Mitochondrial Proteins; Animals; Protein Processing, Post-Translational; Heat-Shock Proteins; Protein Folding
PubMed: 38763405
DOI: 10.1016/j.cstres.2024.05.002 -
Journal of Chemical Information and... May 2024The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the...
The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the β-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the β-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the β-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the β-switch conformation.
Topics: Molecular Dynamics Simulation; Peptides, Cyclic; Protein Binding; Molecular Docking Simulation; Platelet Glycoprotein GPIb-IX Complex; Protein Conformation; von Willebrand Factor; Humans; Binding Sites
PubMed: 38751042
DOI: 10.1021/acs.jcim.4c00100 -
Vaccine Jun 2024The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome....
The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome. Currently, there is no specific treatment or vaccine approved to combat this disease, and thus, developing safe and effective vaccines is a relevant goal. In this study, a multi-epitope protein called rpZDIII was designed based on a series of ZIKV antigenic sequences, a bacterial carrier, and linkers. The analysis of the predicted 3D structure of the rpZDIII chimeric antigen was performed on the AlphaFold 2 server, and it was produced in E. coli and purified from inclusion bodies, followed by solubilization and refolding processes. The yield achieved for rpZDIII was 11 mg/L in terms of pure soluble recombinant protein per liter of fermentation. rpZDIII was deemed immunogenic since it induced serum IgG and IgM responses in mice upon subcutaneous immunization in a three-dose scheme. Moreover, sera from mice immunized with rpZDIII showed neutralizing activity against ZIKV. Therefore, this study reveals rpZDIII as a promising immunogen for the development of a rationally designed multi-epitope vaccine against ZIKV, and completion of its preclinical evaluation is guaranteed.
Topics: Animals; Zika Virus; Antibodies, Neutralizing; Mice; Antibodies, Viral; Zika Virus Infection; Antigens, Viral; Viral Vaccines; Epitopes; Immunoglobulin G; Female; Escherichia coli; Immunoglobulin M; Mice, Inbred BALB C
PubMed: 38749821
DOI: 10.1016/j.vaccine.2024.04.080 -
The Journal of Physical Chemistry. B May 2024In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex...
In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short β-hairpin peptide previously used as a model to study conformational changes in β-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's β-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in β-sheet content at alkaline pH. The β-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the β-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 μs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.
Topics: Hydrogen-Ion Concentration; Kinetics; Protein Folding; Molecular Dynamics Simulation; Oligopeptides; Protein Structure, Secondary
PubMed: 38733339
DOI: 10.1021/acs.jpcb.3c08271 -
Biotechnology Journal May 2024Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has...
Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.
Topics: Humans; Protein Disulfide-Isomerases; Escherichia coli; Interleukin-3; Recombinant Fusion Proteins; Maltose-Binding Proteins; Cell Line, Tumor; Solubility
PubMed: 38719587
DOI: 10.1002/biot.202300581 -
Quarterly Reviews of Biophysics May 2024Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding,... (Review)
Review
Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.
Topics: Animals; Humans; Molecular Motor Proteins; Protein Unfolding; Enzymes; Energy Metabolism
PubMed: 38715547
DOI: 10.1017/S0033583524000052 -
Proceedings of the National Academy of... May 2024Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these...
Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.
Topics: Nanoparticles; Molecular Chaperones; Polymers; Protein Denaturation; Protein Refolding; Protein Folding; Cytochromes c; Laccase; Lipase
PubMed: 38691587
DOI: 10.1073/pnas.2403049121 -
Angewandte Chemie (International Ed. in... Apr 2024Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but...
Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC 17 nM), and their potent activation (EC 56 pM) or inhibition (IC 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.
PubMed: 38679861
DOI: 10.1002/anie.202400632 -
Biomolecules Apr 2024Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority.... (Review)
Review
Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.
Topics: Mycobacterium tuberculosis; Antigens, Bacterial; Virulence Factors; Humans; Tuberculosis Vaccines; Bacterial Proteins; Tuberculosis; Animals; Molecular Chaperones
PubMed: 38672487
DOI: 10.3390/biom14040471 -
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