A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein.

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T-cell activity correlates within individuals, suggesting N antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested

Mechanisms of TRIM21-directed intracellular degradation

The cytosolic antibody receptor TRIM21 has been studied for more than a decade for its role in mediating antibody-dependent neutralization of nonenveloped viruses. The presence of antibodies in the cytosol is abnormal—by binding them and initiating inflammatory signaling cascades, TRIM21 acts as a danger sensor and intracellular effector of humoral immunity. In addition to antibody binding, TRIM21 is a ubiquitin E3 ligase, an enzyme which catalyzes the formation of polymers of the protein ubiquitin around target substrates. When viruses coated in non-neutralizing antibodies escape into the cytosol, TRIM21 binds and decorates the complex with these polyubiquitin chains. Polyubiquitin is a highly-conserved protein post- translational modification with a number of important roles, including the initiation of degradative processes and antiviral genetic programs. Polyubiquitin-coated TRIM21:antibody:virus complexes are degraded by the cell, thus neutralizing viral infection. Its ability to bind generic immunoglobulins has allowed TRIM21 to be developed into the targeted protein degradation technology Trim-Away, whose intracellular mechanism is thought to mirror that of antibody-coated virions. Prior to the work presented here, the specific mechanistic steps following TRIM21-antibody binding had been established in some detail, and were believed to involve the sequential action of ubiquitin E2 conjugating enzymes UBE2W and UBE2N, which cooperated with TRIM21 to catalyze the formation of a specific type of linear polyubiquitin chain linked through lysine 63 on ubiquitin. This was thought to lead to the recruitment of the segregase p97/VCP and the 26S proteosome which together disassembled and degraded the encapsidated virion. In this thesis, I describe my efforts to define the sequential enzymatic mechanism of TRIM21 degradative signaling in greater detail. I report data from a series of biochemical and in-cell experiments which are not consistent with a role for UBE2W in polyubiquitin chain priming on TRIM21 during adenoviral neutralization. Then, I detail my development of two proximity-proteomics screens and a whole-genome CRISPR/Cas9 knockout screen for TRIM21 interacting partners, in both Trim-Away and adenovirus neutralization contexts. Collectively, the data from these experiments led me to hypothesize that the TRIM21 mechanism is plastic, and sensitive to substrate features and intracellular context. The whole-genome screen and follow up validatory experiments were consistent with a new model for TRIM21-adenoviral neutralization—rather than processing through the proteasome, intracellular adenoviruses coated with antibodies are degraded by selective autophagy following polyubiquitin deposition by TRIM21. Taken together, this work represents a clear advancement in understanding how TRIM21 targets substrates for proteolysis, and opens several new lines of inquiry into how the protein affects adaptive immunity at the cellular level.MRC Laboratory of Molecular Biology Cambridge Trus

Rapid Non-Enzymatic Glycation of the Insulin Receptor under Hyperglycemic Conditions Inhibits Insulin Binding In Vitro: Implications for Insulin Resistance

The causes of insulin resistance are not well-understood in either type 1 or type 2 diabetes. Insulin (INS) is known to undergo rapid non-enzymatic covalent conjugation to glucose or other sugars (glycation). Because the insulin receptor (IR) has INS-like regions associated with both glucose and INS binding, we hypothesize that hyperglycemic conditions may rapidly glycate the IR, chronically interfering with INS binding. IR peptides were synthesized spanning IR- associated INS-binding regions. Glycation rates of peptides under hyperglycemic conditions were followed over six days using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. INS conjugated to horse-radish peroxidase was used to determine INS binding to IR peptides in glycated and non-glycated forms. Several IR peptides were glycated up to 14% within days of exposure to 20–60 mM glucose. Rates of IR-peptide glycation were comparable to those of insulin. Glycation of four IR peptides significantly inhibits INS binding to them. Glycation of intact IR also decreases INS binding by about a third, although it was not possible to confirm the glycation sites on the intact IR. Glycation of the IR may therefore provide a mechanism by which INS resistance develops in diabetes. Demonstration of glycation of intact IR in vivo is needed

“Sea Water” Supplemented with Calcium Phosphate and Magnesium Sulfate in a Long-Term Miller-Type Experiment Yields Sugars, Nucleic Acids Bases, Nucleosides, Lipids, Amino Acids, and Oligopeptides

The standard approach to exploring prebiotic chemistry is to use a small number of highly purified reactants and to attempt to optimize the conditions required to produce a particular end product. However, purified reactants do not exist in nature. We have previously proposed that what drives prebiotic evolution are complex chemical ecologies. Therefore, we have begun to explore what happens if one substitutes “sea water”, with its complex mix of minerals and salts, for distilled water in the classic Miller experiment. We have also adapted the apparatus to permit it to be regassed at regular intervals so as to maintain a relatively constant supply of methane, hydrogen, and ammonia. The “sea water” used in the experiments was created from Mediterranean Sea salt with the addition of calcium phosphate and magnesium sulfate. Tests included several types of mass spectrometry, an ATP-monitoring device capable of measuring femtomoles of ATP, and a high-sensitivity cAMP enzyme-linked immunoadsorption assay. As expected, amino acids appeared within a few days of the start of the experiment and accumulated thereafter. Sugars, including glucose and ribose, followed as did long-chain fatty acids (up to C20). At three-to-five weeks after starting the experiment, ATP was repeatedly detected. Thus, we have shown that it is possible to produce a “one-pot synthesis” of most of the key chemical prerequisites for living systems within weeks by mimicking more closely the complexity of real-world chemical ecologies

"Sea Water" Supplemented with Calcium Phosphate and Magnesium Sulfate in a Long-Term Miller-Type Experiment Yields Sugars, Nucleic Acids Bases, Nucleosides, Lipids, Amino Acids, and Oligopeptides.

The standard approach to exploring prebiotic chemistry is to use a small number of highly purified reactants and to attempt to optimize the conditions required to produce a particular end product. However, purified reactants do not exist in nature. We have previously proposed that what drives prebiotic evolution are complex chemical ecologies. Therefore, we have begun to explore what happens if one substitutes "sea water", with its complex mix of minerals and salts, for distilled water in the classic Miller experiment. We have also adapted the apparatus to permit it to be regassed at regular intervals so as to maintain a relatively constant supply of methane, hydrogen, and ammonia. The "sea water" used in the experiments was created from Mediterranean Sea salt with the addition of calcium phosphate and magnesium sulfate. Tests included several types of mass spectrometry, an ATP-monitoring device capable of measuring femtomoles of ATP, and a high-sensitivity cAMP enzyme-linked immunoadsorption assay. As expected, amino acids appeared within a few days of the start of the experiment and accumulated thereafter. Sugars, including glucose and ribose, followed as did long-chain fatty acids (up to C20). At three-to-five weeks after starting the experiment, ATP was repeatedly detected. Thus, we have shown that it is possible to produce a "one-pot synthesis" of most of the key chemical prerequisites for living systems within weeks by mimicking more closely the complexity of real-world chemical ecologies

"Sea Water" Supplemented with Calcium Phosphate and Magnesium Sulfate in a Long-Term Miller-Type Experiment Yields Sugars, Nucleic Acids Bases, Nucleosides, Lipids, Amino Acids, and Oligopeptides.

Peer reviewed: TrueThe standard approach to exploring prebiotic chemistry is to use a small number of highly purified reactants and to attempt to optimize the conditions required to produce a particular end product. However, purified reactants do not exist in nature. We have previously proposed that what drives prebiotic evolution are complex chemical ecologies. Therefore, we have begun to explore what happens if one substitutes "sea water", with its complex mix of minerals and salts, for distilled water in the classic Miller experiment. We have also adapted the apparatus to permit it to be regassed at regular intervals so as to maintain a relatively constant supply of methane, hydrogen, and ammonia. The "sea water" used in the experiments was created from Mediterranean Sea salt with the addition of calcium phosphate and magnesium sulfate. Tests included several types of mass spectrometry, an ATP-monitoring device capable of measuring femtomoles of ATP, and a high-sensitivity cAMP enzyme-linked immunoadsorption assay. As expected, amino acids appeared within a few days of the start of the experiment and accumulated thereafter. Sugars, including glucose and ribose, followed as did long-chain fatty acids (up to C20). At three-to-five weeks after starting the experiment, ATP was repeatedly detected. Thus, we have shown that it is possible to produce a "one-pot synthesis" of most of the key chemical prerequisites for living systems within weeks by mimicking more closely the complexity of real-world chemical ecologies

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

Funder: Boehringer Ingelheim FondsTRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

Acknowledgements: L.K. was supported by a Ph.D. Fellowship from the Boehringer Ingelheim Fonds. This work was supported by the MRC (UK; U105181010), a Wellcome Trust Investigator Award (223054/Z/21/Z) and a Wellcome Trust Collaborator Award (214344/A/18/Z).Funder: Boehringer Ingelheim Fonds (Stiftung für medizinische Grundlagenforschung); doi: https://doi.org/10.13039/501100001645TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates