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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 -
Naunyn-Schmiedeberg's Archives of... Jul 2024Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity...
Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity has been a promising pharmacological strategy for blocking of bladder cancer pathogenesis. BIIB021 is a next-generation HSP90 inhibitor which interrupts ATP hydrolysis process of HSP90 and inhibits the stabilization and correct folding of client proteins. In current study, we aimed to investigate the molecular mechanism of the anticancer activity of BIIB021 in human bladder cancer T24 cells. Our results revealed that nanomolar concentration of BIIB021 decreased viability of T24 cell. BIIB021 downregulated HSP90 expression in T24 cells and inhibited the refolding activity of luciferase in the presence of T24 cell lysate. PCR array data indicated a significant alteration in transcript levels of cancer-related genes involved in metastases, apoptotic cell death, cell cycle, cellular senescence, DNA damage and repair mechanisms, epithelial-to-mesenchymal transition, hypoxia, telomeres and telomerase, and cancer metabolism pathways in T24 cells. All findings hypothesize that BIIB021 could exhibit as effective HSP90 inhibitor in the future for treatment of bladder cancer patients.
Topics: Humans; HSP90 Heat-Shock Proteins; Urinary Bladder Neoplasms; Cell Line, Tumor; Antineoplastic Agents; Cell Survival; Apoptosis; Gene Expression Regulation, Neoplastic
PubMed: 38240781
DOI: 10.1007/s00210-024-02950-x -
Journal of Biochemistry Mar 2024In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this...
In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this study, we assessed the role of the periplasmic protein CdbC in disulfide bond formation and its involvement in mycomembrane biosynthesis. Purified CdbC efficiently refolded scrambled RNaseA, exhibiting prominent disulfide bond isomerase activity. The transcription of cdbC was decreased in cells grown in the presence of the reductant dithiothreitol (DTT). Moreover, unlike wild-type and cdbC-deleted cells, cdbC-overexpressing (P180-cdbC) cells grown in the presence of DTT exhibited retarded growth, abnormal cell morphology, increased cell surface hydrophobicity and altered mycolic acid composition. P180-cdbC cells cultured in a reducing environment accumulated trehalose monocorynomycolate, indicating mycomembrane deformation. Similarly, a two-hybrid analysis demonstrated the interaction of CdbC with the mycoloyltransferases MytA and MytB. Collectively, our findings suggest that CdbC, a periplasmic disulfide bond isomerase, refolds misfolded MytA and MytB and thereby assists in mycomembrane biosynthesis in cells exposed to oxidative conditions.
Topics: Corynebacterium glutamicum; Protein Disulfide-Isomerases; Oxidative Stress; Oxidation-Reduction; Disulfides; Bacterial Proteins
PubMed: 38227582
DOI: 10.1093/jb/mvae005 -
Bioorganic & Medicinal Chemistry Feb 2024The T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an inhibitory immunoreceptor expressed on lymphocytes...
Synthetic studies on the extracellular domain of the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) using Trt-K solubilizing tags.
The T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an inhibitory immunoreceptor expressed on lymphocytes that serves as a promising target for cancer immunotherapy. In this study, facile synthetic protocols to produce the extracellular domain of TIGIT were investigated for applications of TIGIT in mirror-image screening. During the synthesis via sequential native chemical ligations, we encountered problems with significantly poor solubility of the ligated products. Introducing trityl-type solubilizing auxiliaries, which also functioned as temporary protecting groups for cysteine residues, facilitated a flexible order of ligations and efficient purification protocols. After refolding under appropriate conditions, the synthetic TIGIT showed a sufficient affinity toward its target ligand CD155.
Topics: T-Lymphocytes; Immunoglobulins; Receptors, Immunologic; Immunotherapy; Tyrosine
PubMed: 38219557
DOI: 10.1016/j.bmc.2023.117585 -
Archives of Biochemistry and Biophysics Feb 2024Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells...
Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells and tissues and is considerably responsible for the pathologies of diseases like severe malaria. Hemin's hydrophobic chemical nature and structure allow it to bind several proteins leading to their functional modification. Such modifications in physiologically relevant proteins can have a high impact on various cellular processes. HSPA8 is a chaperone that has a protective role in oxidative stress by aiding protein refolding. Through ATPase activity assays we found that hemin can competitively inhibit ATP hydrolysis by the chaperone HSPA8. Hemin as such does not affect the structural integrity of the protein which is inferred from CD spectroscopy and Gel filtration but it hinders the ATP-dependent foldase function of the chaperone. HSPA8 was not able to cause the refolding of the model protein lysozyme in the presence of hemin. The loss in HSPA8 function was due to competition between hemin and ATP as the chaperone was able to regain the foldase function when the concentration of ATP was gradually increased with hemin present at the inhibitory concentration. In-silico studies to establish the competition for the specific binding site revealed that ATP was unable to replace hemin from the ATP binding pocket of HSPA8 and was forced to form a non-specific and unstable complex. In-vitro isothermal calorimetry revealed that the affinity of ATP for binding to HSPA8 was reduced 22 folds in the presence of hemin. The prevention of HSPA8's cytoprotective function by hemin can be a major factor contributing to the overall cellular damage during hemin accumulation in the case of severe malaria and other hemolytic diseases.
Topics: Humans; Hemin; Molecular Chaperones; Hemolysis; Malaria; Adenosine Triphosphatases; Adenosine Triphosphate; HSC70 Heat-Shock Proteins
PubMed: 38215959
DOI: 10.1016/j.abb.2024.109889 -
Nanoscale Feb 2024Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are...
Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are mechanistically complicated and context dependent, the underlying molecular mechanism is not fully understood. Here, we combined single-molecule magnetic tweezers and molecular dynamics (MD) simulation to explore the mechanical role of osmolytes from two different classes, trimethylamine -oxide (TMAO) and trehalose, as mechanical stabilizers of protein structure. We observed that these osmolytes increase the protein L mechanical stability by decreasing unfolding kinetics while accelerating the refolding kinetics under force, eventually shifting the energy landscape toward the folded state. These osmolytes mechanically stabilize the protein L and plausibly guide them to more thermodynamically robust states. Finally, we observed that osmolyte-modulated protein folding increases mechanical work output up to twofold, allowing the protein to fold under a higher force regime and providing a significant implication for folding-induced structural stability in proteins.
Topics: Proteins; Protein Folding; Molecular Dynamics Simulation; Protein Stability; Methylamines; Thermodynamics
PubMed: 38214221
DOI: 10.1039/d3nr00398a -
Redox Biology Feb 2024The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in...
The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.
Topics: Humans; Protein Serine-Threonine Kinases; Pyruvaldehyde; Endothelial Cells; Endoribonucleases; Unfolded Protein Response; Endoplasmic Reticulum Stress; Endoplasmic Reticulum; Glucose
PubMed: 38199038
DOI: 10.1016/j.redox.2024.103025 -
International Immunopharmacology Feb 2024Cat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in...
BACKGROUND
Cat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy.
METHODS
We first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA).
RESULTS
Among several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1.
CONCLUSION
We firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy.
Topics: Humans; Immunoglobulin E; Amino Acid Sequence; Artificial Intelligence; Hypersensitivity; Allergens
PubMed: 38185034
DOI: 10.1016/j.intimp.2024.111488 -
Journal of Integrative Neuroscience Oct 2023Fluctuations in mechanical force vectors within living cells can substantially influence the behavior and functions of proteins. Tau protein can spontaneously be...
Fluctuations in mechanical force vectors within living cells can substantially influence the behavior and functions of proteins. Tau protein can spontaneously be raptured and entangled in refolding under picoNewton compressive forces that are biologically available in a living cell: a hidden aggregation pathway due to stress and crowding. Our findings were achieved through a customized modification of atomic force microscopy (AFM) for single-molecule manipulation. This previously hidden phenomenon of proteins rupturing collectively while subsequently and spontaneously refolding into a complex entangled conformation, distinct from the Tau protein's folded or unfolded states, could potentially explain the early-event initiation of the aggregation of the Tau protein seen in various neurodegenerative diseases. This article introduces our recent discovery of the missing Tau protein property that is of significant relevance to the Tau protein and neurodegenerative disease research and medical treatment, aiming to stimulate the collective observation and a new perspective on the Tau aggregation mechanism and disease mechanism studies.
Topics: Humans; tau Proteins; Protein Aggregates; Neurodegenerative Diseases; Proteins
PubMed: 38176940
DOI: 10.31083/j.jin2206145 -
Protein Science : a Publication of the... Feb 2024Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as...
Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.
Topics: Green Fluorescent Proteins; Biomarkers
PubMed: 38151801
DOI: 10.1002/pro.4886