scholarly journals Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253364
Author(s):  
Lee A. Armstrong ◽  
Sven M. Lange ◽  
Virginia Dee Cesare ◽  
Stephen P. Matthews ◽  
Raja Sekhar Nirujogi ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Transmembrane Protein ◽  
Cross Reactivity ◽  
Alternative Methods ◽  
Protease Domain ◽  
Host Cell Proteins ◽  
Active Protease ◽  
Micromolar Range

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

scholarly journals Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

2020 ◽  
Author(s):  
Lee A. Armstrong ◽  
Sven M. Lange ◽  
Virginia de Cesare ◽  
Stephen P. Matthews ◽  
Raja Sekar Nirujogi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Transmembrane Protein ◽  
Cross Reactivity ◽  
Host Cells ◽  
Alternative Methods ◽  
Structural Proteins ◽  
Active Protease ◽  
Micromolar Range

AbstractOf the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

scholarly journals Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA

2018 ◽  
Vol 76 (4) ◽  
Author(s):  
Kwok-ho Lam ◽  
Stefan Sikorra ◽  
Jasmin Weisemann ◽  
Hannah Maatsch ◽  
Kay Perry ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Biochemical Characterization ◽  
Protease Domain

scholarly journals Thrombin A-Chain: Activation Remnant or Allosteric Effector?

Thrombosis ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Isis S. R. Carter ◽  
Amanda L. Vanden Hoek ◽  
Edward L. G. Pryzdial ◽  
Ross T. A. MacGillivray
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Current Data ◽  
Enzymatic Reactions ◽  
Allosteric Effector ◽  
Naturally Occurring ◽  
Physiologic Function ◽  
A Chain

Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain.

scholarly journals A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

2020 ◽  
Vol 295 (15) ◽  
pp. 5051-5066 ◽  
Author(s):  
Wei Tan ◽  
Tian-Hua Liao ◽  
Jin Wang ◽  
Yu Ye ◽  
Yu-Chen Wei ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Inorganic Nitrogen ◽  
Nitrate Assimilation ◽  
Nitrogen Sources ◽  
Electron Transfer Mechanism ◽  
Cofactor Binding ◽  
Environmental Mycobacteria

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN–FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives

2008 ◽  
Vol 100 (05) ◽  
pp. 920-928 ◽  
Author(s):  
Henrik Østergaard ◽  
Robert J. Bayer ◽  
Matt S. Kalo ◽  
Kyle Kinealy ◽  
Pernille K. Holm ◽  
...  
Keyword(s):  
Half Life ◽  
Biochemical Characterization ◽  
Factor Viia ◽  
Protease Domain ◽  
Limited Effect ◽  
Bleeding Episodes ◽  
Coagulation Pathway ◽  
Inhibitor Patient

SummaryProphylaxis with 2–4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient,recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface,TF,FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylation™ technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.

Sign in / Sign up

Export Citation Format

Share Document