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Toxins May 2021The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb) mediates internalization... (Review)
Review
The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin-receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing infection. The Gb levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb is upregulated in the neovasculature of many tumours, irrespective of tumour Gb status. Gb is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.
Topics: Animals; Bacterial Toxins; Cholera Toxin; Endoplasmic Reticulum; Gaucher Disease; Humans; Protein Subunits; Proteostasis Deficiencies; Shiga Toxins; Trihexosylceramides
PubMed: 34073185
DOI: 10.3390/toxins13060378 -
Science (New York, N.Y.) Oct 2022Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we...
Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we use deep network hallucination to generate a wide range of symmetric protein homo-oligomers given only a specification of the number of protomers and the protomer length. Crystal structures of seven designs are very similar to the computational models (median root mean square deviation: 0.6 angstroms), as are three cryo-electron microscopy structures of giant 10-nanometer rings with up to 1550 residues and symmetry; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be generated using deep learning and pave the way for the design of increasingly complex components for nanomachines and biomaterials.
Topics: Biocompatible Materials; Cryoelectron Microscopy; Deep Learning; Models, Molecular; Protein Conformation; Protein Engineering; Protein Subunits
PubMed: 36108048
DOI: 10.1126/science.add1964 -
Cell Sep 2020Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the...
Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1 mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1 mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase β subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.
Topics: Adenosine Triphosphate; Animals; Cell Line; Citric Acid Cycle; Fibroblasts; Fragile X Mental Retardation Protein; Fragile X Syndrome; HEK293 Cells; Humans; Mice; Neurons; Protein Subunits; RNA, Messenger; Synapses
PubMed: 32795412
DOI: 10.1016/j.cell.2020.07.008 -
The Journal of General Physiology Oct 2022The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and...
The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ βENaC ≫ αENaC in both organs. Transcript abundance in the kidney, measured with digital-drop PCR and RNAseq, was similar for the three subunits. In both organs, the calculated protein expression of all subunits was much larger than that required to account for maximal Na+ currents measured in these cells, implying a large excess of subunit protein. Whole-kidney biotinylation indicated that at least 5% of β and γ subunits in the kidney and 3% in the colon were expressed on the surface under conditions of salt restriction, which maximizes ENaC-dependent Na+ transport. This indicates a 10- to 100-fold excess of βENaC and γENaC subunits at the surface relative to the requirement for channel activity. We conclude that these epithelia make much more ENaC protein than is required for the physiological function of the channel. This could facilitate rapid regulation of the channels at the cell surface by insuring a large population of inactive, recruitable subunits.
Topics: Animals; Cell Membrane; Epithelial Sodium Channels; Epithelium; Kidney; Protein Subunits; Rats; Sodium
PubMed: 35939271
DOI: 10.1085/jgp.202213124 -
The FEBS Journal Feb 2021Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through... (Review)
Review
Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through dephosphorylation of proteins, which have been phosphorylated by protein kinases. A large understanding of their working has been sourced from animal systems rather than the plant or the prokaryotic systems. The eukaryotic protein phosphatases include phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine(Ser)/threonine(Thr)-specific phosphatases (STPs), while PTP family is Tyr specific. Dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. PTPs lack sequence homology with STPs, indicating a difference in catalytic mechanisms, while the PPP and PPM families share a similar structural fold indicating a common catalytic mechanism. The catalytic cysteine (Cys) residue in the conserved HCX R active site motif of the PTPs acts as a nucleophile during hydrolysis. The PPP members require metal ions, which coordinate the phosphate group of the substrate, followed by a nucleophilic attack by a water molecule and hydrolysis. The variable holoenzyme assembly of protein phosphatase(s) and the overlap with other post-translational modifications like acetylation and ubiquitination add to their complexity. Though their functional characterization is extensively reported in plants, the mechanistic nature of their action is still being explored by researchers. In this review, we exclusively overview the plant protein phosphatases with an emphasis on their mechanistic action as well as structural characteristics.
Topics: Biocatalysis; Catalytic Domain; Models, Molecular; Phosphoprotein Phosphatases; Phosphorylation; Plant Proteins; Protein Domains; Protein Subunits; Signal Transduction; Substrate Specificity
PubMed: 32542989
DOI: 10.1111/febs.15454 -
The Journal of Biological Chemistry Dec 2022Protein phosphatase 2A (PP2A) is a family of serine threonine phosphatases responsible for regulating protein phosphorylation, thus opposing the activity of cellular... (Review)
Review
Protein phosphatase 2A (PP2A) is a family of serine threonine phosphatases responsible for regulating protein phosphorylation, thus opposing the activity of cellular kinases. PP2A is composed of a catalytic subunit (PP2A Cα/β) and scaffolding subunit (PP2A Aα/β) and various substrate-directing B regulatory subunits. PP2A biogenesis is regulated at multiple levels. For example, the sequestration of the free catalytic subunit during the process of biogenesis avoids promiscuous phosphatase activity. Posttranslational modifications of PP2A C direct PP2A heterotrimeric formation. Additionally, PP2A functions as a haploinsufficient tumor suppressor, where attenuated PP2A enzymatic activity creates a permissive environment for oncogenic transformation. Recent work studying PP2A in cancer showed that its role in tumorigenesis is more nuanced, with some holoenzymes being tumor suppressive, while others are required for oncogenic transformation. In cancer biology, PP2A function is modulated through various mechanisms including the displacement of specific B regulatory subunits by DNA tumor viral antigens, by recurrent mutations, and through loss of carboxymethyl-sensitive heterotrimeric complexes. In aggregate, these alterations bias PP2A activity away from its tumor suppressive functions and toward oncogenic ones. From a therapeutic perspective, molecular glues and disruptors present opportunities for both the selective stabilization of tumor-suppressive holoenzymes and disruption of holoenzymes that are pro-oncogenic. Collectively, these approaches represent an attractive cancer therapy for a wide range of tumor types. This review will discuss the mechanisms by which PP2A holoenzyme formation is dysregulated in cancer and the current therapies that are aimed at biasing heterotrimer formation of PP2A for the treatment of cancer.
Topics: Humans; Holoenzymes; Neoplasms; Phosphorylation; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Subunits
PubMed: 36328247
DOI: 10.1016/j.jbc.2022.102656 -
Bioorganic Chemistry Mar 2023The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for...
The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.
Topics: Alkenes; Protein Subunits; Heterocyclic Compounds; Amino Acids; Cycloaddition Reaction
PubMed: 36642019
DOI: 10.1016/j.bioorg.2023.106359 -
Frontiers in Endocrinology 2021The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific β subunit. They act by activating specific leucine-rich repeat G... (Review)
Review
The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific β subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin β subunit (βhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) β subunit is also reported to have biological effects. The GPH α- and β subunits are derived from precursor genes ( and , respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs ( and , respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.
Topics: Amino Acid Sequence; Animals; Glycoprotein Hormones, alpha Subunit; Glycoproteins; Humans; Peptide Hormones; Protein Subunits; Receptors, G-Protein-Coupled
PubMed: 34671318
DOI: 10.3389/fendo.2021.731966 -
Biomolecules Apr 2020Asparagine-linked glycosylation, also known as -linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all... (Review)
Review
Asparagine-linked glycosylation, also known as -linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell-cell communication, and stability. Defects in -linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). -linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of -linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon , and the PglB protein of the bacterium are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the -linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in -linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of -linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the -linked glycosylation process in eukaryotes poses an important future goal.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Glycosylation; Hexosyltransferases; Humans; Isoenzymes; Membrane Proteins; Models, Molecular; Protein Subunits
PubMed: 32316603
DOI: 10.3390/biom10040624 -
Scientific Reports Feb 2021The worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as...
The worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. Existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. Here, we are using in silico modelling of possible SARS-CoV-2 drug targets, as deposited on the Protein Databank (PDB), and ascertain their dynamics, flexibility and rigidity. For example, for the SARS-CoV-2 spike protein-using its complete homo-trimer configuration with 2905 residues-our method identifies a large-scale opening and closing of the S1 subunit through movement of the S[Formula: see text] domain. We compute the full structural information of this process, allowing for docking studies with possible drug structures. In a dedicated database, we present similarly detailed results for the further, nearly 300, thus far resolved SARS-CoV-2-related protein structures in the PDB.
Topics: Antiviral Agents; Binding Sites; COVID-19; Crystallography, X-Ray; Drug Development; Humans; Models, Molecular; Pandemics; Protein Binding; Protein Domains; Protein Multimerization; Protein Subunits; SARS-CoV-2; Spike Glycoprotein, Coronavirus; COVID-19 Drug Treatment
PubMed: 33608565
DOI: 10.1038/s41598-021-82849-2