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Progress in Molecular Biology and... 2024In order for an ordered protein to perform its specific function, it must have a specific molecular structure. Information about this structure is encoded in the... (Review)
Review
In order for an ordered protein to perform its specific function, it must have a specific molecular structure. Information about this structure is encoded in the protein's amino acid sequence. The unique functional state is achieved as a result of a specific process, known as protein folding. However, as a result of partial or complete unfolding of the polypeptide chain, proteins may misfold and aggregate, leading to the formation of various aggregated structures, such as like amyloid aggregates with the cross-β structure. A variety of cellular biological processes can be affected by protein aggregates that consume essential factors necessary for maintaining proteostasis, which leads to the proteostasis imbalance and further accumulation of protein aggregates, often resulting in age-related neurodegenerative disease progression and aging. However, in addition to their well-established pathological effects, amyloids also play various physiological roles, and many important biological processes involve such 'functional amyloids'. This chapter represents a brief overview of the protein aggregation phenomenon outlines a timeline provides of some key discoveries in this exciting field.
Topics: Humans; Protein Aggregates; Animals; Amyloid; Protein Aggregation, Pathological; Protein Folding; Proteins
PubMed: 38811077
DOI: 10.1016/bs.pmbts.2024.03.007 -
Metabolism: Clinical and Experimental Aug 2023Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are...
OBJECTIVE
Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease.
METHODS
We first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed.
RESULTS
We found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1 mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion.
CONCLUSION
Our findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.
Topics: Animals; Mice; Bile Acids and Salts; Cholesterol; Gallstones; Glutaredoxins; Lipid Metabolism; Liver; Mice, Inbred C57BL; Protein S; Proteomics
PubMed: 37277061
DOI: 10.1016/j.metabol.2023.155610 -
International Journal of Molecular... Jul 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E),...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50-200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors (a dimensional volume) suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target (protein, antibody, ligand). The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space.
Topics: Humans; SARS-CoV-2; Spike Glycoprotein, Coronavirus; COVID-19; Angiotensin-Converting Enzyme 2; Receptors, Virus; Protein Binding; Ligands; Peptidyl-Dipeptidase A
PubMed: 37569436
DOI: 10.3390/ijms241512058 -
Nature Structural & Molecular Biology Sep 2023Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear....
Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.
Topics: Humans; Animals; Rabbits; Saccharomyces cerevisiae; Mammals; Ribosomes; Ubiquitination; Mass Spectrometry; Transcription Factors; Receptors, CCR4; Ribonucleases; Saccharomyces cerevisiae Proteins
PubMed: 37653243
DOI: 10.1038/s41594-023-01075-8 -
Nature Protocols Sep 2023Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many... (Review)
Review
Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.
Topics: Animals; Caenorhabditis elegans; Animals, Genetically Modified; Membrane Proteins; Chromatography, Gel; Cell Line; Caenorhabditis elegans Proteins
PubMed: 37495753
DOI: 10.1038/s41596-023-00852-5 -
Cell Reports Nov 2023In this study, we investigate the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the...
In this study, we investigate the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear. We reveal that in Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. We reveal taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. We further show that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, our results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception.
Topics: Animals; Taste; Taste Perception; Drosophila; Sugars; Hedgehog Proteins; Carbohydrates; Drosophila Proteins; Adipose Tissue; Fatty Acids; Drosophila melanogaster
PubMed: 37934669
DOI: 10.1016/j.celrep.2023.113387 -
Journal of Proteome Research Jul 2023-Palmitoylation is the covalent attachment of C14:0-C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly...
-Palmitoylation is the covalent attachment of C14:0-C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly abundant in neurons, where it plays a role in neuronal development and is implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The knowledge of -palmitoylation in neurodevelopment is limited due to technological challenges in analyzing this highly hydrophobic protein modification. Here, we used two orthogonal methods, acyl-biotin exchange (ABE) and lipid metabolic labeling (LML), to identify -palmitoylated proteins and sites during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. We identified 2002 putative -palmitoylated proteins in total, of which 650 were found with both methods. Significant changes in the abundance of -palmitoylated proteins were detected, in particular for several processes and protein classes that are known to be important for neuronal differentiation, which include proto-oncogene tyrosine-protein kinase receptor (RET) signal transduction, SNARE protein-mediated exocytosis, and neural cell adhesion molecules. Overall, -palmitoylation profiling by employing ABE and LML in parallel during RA-induced differentiation of SH-SY5Y cells revealed a subset of high confidence bona fide -palmitoylated proteins and suggested an important role for -palmitoylation in neuronal differentiation.
Topics: Humans; Tretinoin; Lipoylation; Neuroblastoma; Cell Differentiation; Proteins; Lipids; Cell Line, Tumor
PubMed: 37294931
DOI: 10.1021/acs.jproteome.3c00151 -
The EMBO Journal Oct 2023The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly...
The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1 phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.
Topics: Autophagy; Autophagy-Related Proteins; Phagosomes; Phosphorylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37635626
DOI: 10.15252/embj.2022112814 -
Journal of Structural Biology Sep 2023Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a...
Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.
Topics: Humans; Adenosine Triphosphate; Binding Sites; Cell Cycle Proteins; Eukaryotic Initiation Factor-2; Mental Retardation, X-Linked; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37507029
DOI: 10.1016/j.jsb.2023.108006 -
Microbial Biotechnology Nov 2023Iron is an essential element for all eukaryote organisms because of its redox properties, which are important for many biological processes such as DNA synthesis,... (Review)
Review
Iron is an essential element for all eukaryote organisms because of its redox properties, which are important for many biological processes such as DNA synthesis, mitochondrial respiration, oxygen transport, lipid, and carbon metabolism. For this reason, living organisms have developed different strategies and mechanisms to optimally regulate iron acquisition, transport, storage, and uptake in different environmental responses. Moreover, iron plays an essential role during microbial infections. Saccharomyces cerevisiae has been of key importance for decrypting iron homeostasis and regulation mechanisms in eukaryotes. Specifically, the transcription factors Aft1/Aft2 and Yap5 regulate the expression of genes to control iron metabolism in response to its deficiency or excess, adapting to the cell's iron requirements and its availability in the environment. We also review which iron-related virulence factors have the most common fungal human pathogens (Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans). These factors are essential for adaptation in different host niches during pathogenesis, including different fungal-specific iron-uptake mechanisms. While being necessary for virulence, they provide hope for developing novel antifungal treatments, which are currently scarce and usually toxic for patients. In this review, we provide a compilation of the current knowledge about the metabolic response to iron deficiency and excess in fungi.
Topics: Humans; Transcription Factors; Iron Deficiencies; Iron; Saccharomyces cerevisiae; Biological Transport; Gene Expression Regulation, Fungal; Trans-Activators; Saccharomyces cerevisiae Proteins; Basic-Leucine Zipper Transcription Factors
PubMed: 37804207
DOI: 10.1111/1751-7915.14346