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1. Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

   https://doi.org/10.1038/s42004-021-00476-4  

   Conti, Brian J. ; Leicht, Andrew S. ; Kirchdoerfer, Robert N. ; Sussman, Michael R. ( March 2021 , Communications Chemistry)

   Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of thenonstructuralprotein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

    

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2. Mass spectrometry‐based proteomic platforms for better understanding of SARS‐CoV‐2 induced pathogenesis and potential diagnostic approaches

   https://doi.org/10.1002/pmic.202000279  

   Ahsan, Nagib ; Rao, R. Shyama Prasad ; Wilson, Rashaun S. ; Punyamurtula, Ujwal ; Salvato, Fernanda ; Petersen, Max ; Ahmed, Mohammad Kabir ; Abid, M. Ruhul ; Verburgt, Jacob C. ; Kihara, Daisuke ; et al ( May 2021 , PROTEOMICS)

   While protein–protein interaction is the first step of the SARS‐CoV‐2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)‐based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS‐CoV‐2‐mediated infections in humans. Comparative analysis of cell‐lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS‐CoV‐2 infection is still incomplete and the tissue‐specific response to SARS‐CoV‐2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross‐comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K‐Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS‐CoV‐2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS‐CoV‐2 responsive age‐, gender‐dependent, tissue‐specific protein targets.

    

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3. Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection

   https://doi.org/10.1038/s41598-023-29119-5  

   Aryal, Baikuntha ; Tillotson, Joseph ; Ok, Kiwon ; Stoltzfus, Andrew T. ; Michel, Sarah L. J. ; Rao, V. Ashutosh ( February 2023 , Scientific Reports)

   Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.

    

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4. Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins

   https://doi.org/10.3389/fchem.2022.1017394  

   Iyengar, Suhasini M. ; Barnsley, Kelly K. ; Vu, Hoang Yen ; Bongalonta, Ian Jef ; Herrod, Alyssa S. ; Scott, Jasmine A. ; Ondrechen, Mary Jo ( October 2022 , Frontiers in Chemistry)

   Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2′-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145. For the RNA methyltransferase, the catalytic lysine K6968 that serves as a Brønsted base has significant population of its deprotonated state via strong coupling with K6844 and Y6845. For the main protease, Partial Order Optimum Likelihood (POOL) predicts two clusters of biochemically active residues; one includes the catalytic H41 and C145 and neighboring residues. The other surrounds a second pocket adjacent to the catalytic site and includes S1 residues F140, L141, H163, E166, and H172 and also S2 residue D187. This secondary recognition site could serve as an alternative target for the design of molecular probes. From in silico screening of library compounds, ligands with predicted affinity for the secondary site are reported. For the NSP16-NSP10 complex that comprises the RNA methyltransferase, three different sites are predicted. One is the catalytic core at the conserved K-D-K-E motif that includes catalytic residues D6928, K6968, and E7001 plus K6844. The second site surrounds the catalytic core and consists of Y6845, C6849, I6866, H6867, F6868, V6894, D6895, D6897, I6926, S6927, Y6930, and K6935. The third is located at the heterodimer interface. Ligands predicted to have high affinity for the first or second sites are reported. Three sites are also predicted for the nucleocapsid protein. This work uncovers key interactions that contribute to the function of the three viral proteins and also suggests alternative sites for ligand design. 

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5. Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins

   Iyengar, Suhasini M. ; Barnsley, Kelly K. ; Vu, Hoang Yen ; Bongalonta, Ian Jef ; Herrod, Alyssa S. ; Scott, Jasmine A. ; Ondrechen, Mary Jo ( October 2022 , Frontiers in chemistry)

   Bienstock, Rachelle J. (Ed.)

   Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2’-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145. For the RNA methyltransferase, the catalytic lysine K6968 that serves as a Brønsted base has significant population of its deprotonated state via strong coupling with K6844 and Y6845. For the main protease, Partial Order Optimum Likelihood (POOL) predicts two clusters of biochemically active residues; one includes the catalytic H41 and C145 and neighboring residues. The other surrounds a second pocket adjacent to the catalytic site and includes S1 residues F140, L141, H163, E166, and H172 and also S2 residue D187. This secondary recognition site could serve as an alternative target for the design of molecular probes. From in silico screening of library compounds, ligands with predicted affinity for the secondary site are reported. For the NSP16-NSP10 complex that comprises the RNA methyltransferase, three different sites are predicted. One is the catalytic core at the conserved K-D-K-E motif that includes catalytic residues D6928, K6968, and E7001 plus K6844. The second site surrounds the catalytic core and consists of Y6845, C6849, I6866, H6867, F6868, V6894, D6895, D6897, I6926, S6927, Y6930, and K6935. The third is located at the heterodimer interface. Ligands predicted to have high affinity for the first or second sites are reported. Three sites are also predicted for the nucleocapsid protein. This work uncovers key interactions that contribute to the function of the three viral proteins and also suggests alternative sites for ligand design. 

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