-
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 -
Cell Stress & Chaperones Sep 2023Chaperone proteins have crucial roles to play in all animal species and are involved in mediating both the folding of newly synthesized peptides into their mature... (Review)
Review
Chaperone proteins have crucial roles to play in all animal species and are involved in mediating both the folding of newly synthesized peptides into their mature conformation, the refolding of misfolded proteins, and the trafficking of proteins between subcellular compartments. These highly conserved proteins have particularly important roles to play in dealing with disruptions of the proteome as a result of environmental stress since abiotic factors, including temperature, pressure, oxygen, water availability, and pollutants can readily disrupt the conformation and/or function of all types of proteins, e.g., enzymes, transporters, and structural proteins. The current review provides an update on recent advances in understanding the roles and responses of chaperones in aiding animals to deal with environmental stress, offering new information on chaperone action in supporting survival strategies including torpor, hibernation, anaerobiosis, estivation, and cold/freeze tolerance among both vertebrate and invertebrate species.
Topics: Animals; Heat-Shock Proteins; Molecular Chaperones; Protein Folding; Cold Temperature; Temperature
PubMed: 36441380
DOI: 10.1007/s12192-022-01312-x -
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 -
Experimental & Molecular Medicine Nov 2023Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling...
Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling and pain-causing properties. ER stress has been reported to be closely related to the progression of OA. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) pathway, which is highly expressed in the chondrocytes of OA patients, promotes the degradation and refolding of abnormal proteins during ER stress and maintains the stability of the ER environment of chondrocytes, but its function and the underlying mechanisms of how it contributes to the progression of OA remain unclear. This study investigates the role of IRE1α/ERN1 in OA. Specific deficiency of ERN1 in chondrocytes spontaneously resulted in OA-like cartilage destruction and accelerated OA progression in a surgically induced arthritis model. Local delivery of AdERN1 relieved degradation of the cartilage matrix and prevented OA development in an ACLT-mediated model. Mechanistically, progranulin (PGRN), an intracellular chaperone, binds to IRE1α, promoting its phosphorylation and splicing of XBP1u to generate XBP1s. XBP1s protects articular cartilage through TNF-α/ERK1/2 signaling and further maintains collagen homeostasis by regulating type II collagen expression. The chondroprotective effect of IRE1α/ERN1 is dependent on PGRN and XBP1s splicing. ERN1 deficiency accelerated cartilage degeneration in OA by reducing PGRN expression and XBP1s splicing, subsequently decreasing collagen II expression and triggering collagen structural abnormalities and an imbalance in collagen homeostasis. This study provides new insights into OA pathogenesis and the UPR and suggests that IRE1α/ERN1 may serve as a potential target for the treatment of joint degenerative diseases, including OA.
Topics: Humans; Protein Serine-Threonine Kinases; Progranulins; Endoribonucleases; Quality of Life; Osteoarthritis; Chondrocytes; Cartilage, Articular; Collagen; Homeostasis; X-Box Binding Protein 1
PubMed: 37907740
DOI: 10.1038/s12276-023-01106-w -
Protein Expression and Purification Aug 2023MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large...
MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.
Topics: Escherichia coli; Matrix Metalloproteinase 2; Recombinant Proteins; Bacterial Proteins; Inclusion Bodies; Protein Folding; Protein Refolding
PubMed: 37094772
DOI: 10.1016/j.pep.2023.106278 -
Journal of Colloid and Interface Science May 2024Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded...
HYPOTHESIS
Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation.
EXPERIMENTS
In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and β-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant β-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein.
FINDINGS
The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation.
PubMed: 38838632
DOI: 10.1016/j.jcis.2024.05.157 -
Cancers Feb 2024Heat shock proteins (HSPs) are developmentally conserved families of protein found in both prokaryotic and eukaryotic organisms. HSPs are engaged in a diverse range of... (Review)
Review
Heat shock proteins (HSPs) are developmentally conserved families of protein found in both prokaryotic and eukaryotic organisms. HSPs are engaged in a diverse range of physiological processes, including molecular chaperone activity to assist the initial protein folding or promote the unfolding and refolding of misfolded intermediates to acquire the normal or native conformation and its translocation and prevent protein aggregation as well as in immunity, apoptosis, and autophagy. These molecular chaperonins are classified into various families according to their molecular size or weight, encompassing small HSPs (e.g., HSP10 and HSP27), HSP40, HSP60, HSP70, HSP90, and the category of large HSPs that include HSP100 and ClpB proteins. The overexpression of HSPs is induced to counteract cell stress at elevated levels in a variety of solid tumors, including anticancer chemotherapy, and is closely related to a worse prognosis and therapeutic resistance to cancer cells. HSPs are also involved in anti-apoptotic properties and are associated with processes of cancer progression and development, such as metastasis, invasion, and cell proliferation. This review outlines the previously mentioned HSPs and their significant involvement in diverse mechanisms of tumor advancement and metastasis, as well as their contribution to identifying potential targets for therapeutic interventions.
PubMed: 38339390
DOI: 10.3390/cancers16030638 -
Methods in Molecular Biology (Clifton,... 2024Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol...
Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol for conducting a protein nanomechanical experiment using AFM, covering both the force-extension and force-clamp modes. Combined, these experiments provide an integrated vista of the molecular mechanisms-and their associated kinetics-underpinning the mechanical unfolding and refolding of individual proteins when exposed to mechanical load.
Topics: Microscopy, Atomic Force; Proteins; Nanotechnology; Mechanical Phenomena; Molecular Conformation; Protein Folding; Protein Unfolding
PubMed: 37824012
DOI: 10.1007/978-1-0716-3377-9_16 -
Journal of Molecular Biology Jul 2024The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the... (Review)
Review
The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70. In this review, we describe the physiological roles of JDPs in the Saccharomyces cerevisiae PQC system, with a focus on the abundant JDP generalists, Zuo1, Ydj1 and Sis1, which function in fundamental cellular processes. Ribosome-bound Zuo1 cooperates with the Hsp70 chaperones Ssb1/2 in folding and assembly of nascent polypeptides. Ydj1 and Sis1 cooperate with the Hsp70 members Ssa1 to Ssa4 to exert overlapping functions in protein folding and targeting of newly synthesized proteins to organelles including mitochondria and facilitating the degradation of aberrant proteins by E3 ligases. Furthermore, they act in protein disaggregation reactions, though Ydj1 and Sis1 differ in their modes of Hsp70 cooperation and substrate specificities. This results in functional specialization as seen in prion propagation and the underlying dominant role of Sis1 in targeting Hsp70 for shearing of prion amyloid fibrils.
Topics: Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; HSP70 Heat-Shock Proteins; Protein Folding; HSP40 Heat-Shock Proteins; Molecular Chaperones; Protein Domains; Heat-Shock Proteins
PubMed: 38331212
DOI: 10.1016/j.jmb.2024.168484 -
Current Issues in Molecular Biology Jan 2024In this study, we review the properties of three anionic detergents, sodium dodecyl sulfate (SDS), Sarkosyl, and sodium lauroylglutamate (SLG), as they play a critical... (Review)
Review
In this study, we review the properties of three anionic detergents, sodium dodecyl sulfate (SDS), Sarkosyl, and sodium lauroylglutamate (SLG), as they play a critical role in molecular biology research. SDS is widely used in electrophoresis and cell lysis for proteomics. Sarkosyl and, more frequently, SDS are used for the characterization of neuropathological protein fibrils and the solubilization of proteins. Many amyloid fibrils are resistant to SDS or Sarkosyl to different degrees and, thus, can be readily isolated from detergent-sensitive proteins. SLG is milder than the above two detergents and has been used in the solubilization and refolding of proteins isolated from inclusion bodies. Here, we show that both Sarkosyl and SLG have been used for protein refolding, that the effects of SLG on the native protein structure are weaker for SLG, and that SLG readily dissociates from the native proteins. We propose that SLG may be effective in cell lysis for functional proteomics due to no or weaker binding of SLG to the native proteins.
PubMed: 38248342
DOI: 10.3390/cimb46010040