A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals DOI Creative Commons

Anahi G. Jimenez-Campos,

Lucas I. Maestas,

Nileena Velappan

et al.

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0309305 - e0309305

Published: Dec. 26, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple fast tests that reliably detect active infections with SARS-CoV-2 in the early stage of infection. Here we introduce rapid activity-based diagnostic (ABDx) test identifies by measuring activity viral enzyme, Papain-Like protease (PLpro). The system consists peptide fluoresces when cleaved SARS PLpro is crude, unprocessed lysates from tongue scrapes saliva. Test results obtained 30 minutes or less using widely available fluorescence plate readers, battery-operated portable instrument on-site testing. Proof-of-concept was study on clinical specimens collected patients COVID-19 like symptoms who tested positive (n = 10) negative LIAT RT-PCR nasal mid turbinate swabs. When saliva these in-house endpoint RT-PCR, 17 were only 5 negative, which became days later. correlated cases (3 out 3 negatives 14 16 positives, one invalid specimen). Despite small number samples, agreement significant (p value 0.01). Two false detected, sample late Ct 35 indicating an infection no longer present. assay easily scalable expected all viable variants, making it attractive as screening surveillance tool. Additionally, show feasibility platform new homogeneous phenotypic antiviral drugs neutralizing antibodies.

Language: Английский

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers DOI Creative Commons
Enrique Álvarez, Michela Falqui, Laura Sin

et al.

Vaccines, Journal Year: 2024, Volume and Issue: 12(2), P. 153 - 153

Published: Feb. 1, 2024

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral cancer-related processes, among others. well-established effects of ISGylation contrast with intriguing dual cancer, exhibiting both suppressive promoting depending on tumour type. multifaceted extend beyond processes signalling, chemotaxis, anti-tumour effects. Moreover, emerges as promising adjuvant vaccine development, enhancing responses against viral antigens demonstrating efficacy cancer models. As therapeutic target treatment, exhibits double-edged nature, or suppressing oncogenesis context. This review aims contribute future studies exploring modulation therapy, potentially paving way for development novel interventions, precision medicine.

Language: Английский

Citations

9
Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases DOI Creative Commons
Zhou Zhao, Rachel O’Dea, Kim Wendrich

et al.

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(38), P. 20801 - 20812

Published: Sept. 15, 2023

Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates these enzymes that is based on activation C-terminal Ub/Ubl hydrazides to acyl azides their subsequent functionalization isopeptides. The procedure complemented native purification routes thus circumvents previous need desulfurization refolding. Its broad applicability was demonstrated generation fully cleavable Ub, SUMO1, SUMO2, NEDD8, ISG15, Fubi. We employed reagents investigation substrate specificities human UCHL3, USPL1, USP2, USP7, USP16, USP18, USP36. Pronounced selectivity USPL1 SUMO2/3 over SUMO1 observed, which rationalize crystal structures biochemical assays, revealing SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, investigated recently identified Fubi proteases USP16 USP36 found both act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Surprisingly, also noticed toward previously not in chemoproteomics, makes first DUBs specific isopeptidase three modifiers. methods described here isopeptide-linked, folded will aid characterization further DUBs/Ubl More broadly, our findings highlight possible limitations associated fluorogenic activity-based probes stress importance isopeptide-containing validating activities quantifying specificities.

Language: Английский

Citations

11
Chemical tools to define and manipulate interferon-inducible Ubl protease USP18 DOI Creative Commons

Griffin J. Davis,

Anthony O. Omole, Ye‐Jin Jung

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 22, 2025

Abstract Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into C-terminal tail ISG15, enabling selective detection USP18 activity over other cross-reactive deubiquitinases (DUBs) such as USP5 USP14. Combined with ubiquitin-based DUB ABP, ABP employed in chemoproteomics screening platform to identify assess inhibitors DUBs including USP18. We further demonstrate ABPs can be utilized profile differential activities lung cancer cell lines, providing strategy will help define activity-related landscape different disease states unravel important (de)ISGylation-dependent biological processes.

Language: Английский

Citations

0
ISGylation: is our genome yearning for such a modification? DOI Creative Commons
Zheng Chen, Zheng Li, Ying Wang

et al.

Acta Biochimica et Biophysica Sinica, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 1, 2025

ISGylation is the post-translational modification of protein substrates covalently conjugated with ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). Although initially linked to antiviral immunity, recent evidence highlights important roles for in various biological processes, such as maintaining genomic stability, promoting tumourigenesis, and being involved other pathological conditions. In this review, we examine molecular mechanisms underlying ISGylation, its interplay modifications, involvement diverse processes. We propose future research directions advance field discuss how might be harnessed ensure human health, particularly genome instability-associated diseases.

Language: Английский

Citations

0
O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression DOI
Jianxin Zhao, Jie Hua,

Yahui Zhan

et al.

Journal of Biological Chemistry, Journal Year: 2024, Volume and Issue: 300(4), P. 107150 - 107150

Published: March 9, 2024

Language: Английский

Citations

2
The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication DOI Creative Commons
Chaohui Lin, Edmund Osei Kuffour,

Taolan Li

et al.

Viruses, Journal Year: 2024, Volume and Issue: 16(4), P. 485 - 485

Published: March 22, 2024

The innate immune response to viruses is formed in part by interferon (IFN)-induced restriction factors, including ISG15, p21, and SAMHD1. IFN production can be blocked the ISG15-specific protease USP18. HIV-1 has evolved circumvent host surveillance. This mechanism might involve In our recent studies, we demonstrate that infection induces USP18, which dramatically enhances replication abrogating antiviral function of p21. USP18 downregulates p21 accumulating misfolded dominant negative p53, inactivates wild-type p53 transactivation, leading upregulation key enzymes involved de novo dNTP biosynthesis pathways inactivated Despite USP18-mediated increase DNA infected cells, it intriguing note cGAS-STING-mediated sensing viral abrogated. Indeed, expression or knockout ISG15 inhibits HIV-1. We STING ISGylated at residues K224, K236, K289, K347, K338, K370. inhibition K289-linked ISGylation suppresses its oligomerization induction. propose human a novel factor potentially contributes multiple ways replication.

Language: Английский

Citations

1
Chemical tools to define and manipulate interferon-inducible Ubl protease USP18 DOI Creative Commons

Griffin J. Davis,

Anthony O. Omole, Ye‐Jin Jung

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 8, 2024

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable selectively detecting USP18 activity over other cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into C-terminal tail ISG15. Combining with ubiquitin-based DUB ABP, selective ABP employed in chemoproteomic screening platform to identify assess inhibitors DUBs including USP18. We further demonstrate that ABPs can be utilized profile differential activities lung cancer cell lines, providing strategy will help define activity-related landscape different disease states unravel important (de)ISGylation-dependent biological processes.

Language: Английский

Citations

0
Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41 DOI Creative Commons

Thomas Bonacci,

Derek L. Bolhuis, Nicholas G. Brown

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 28, 2024

The ubiquitin-like protein ISG15 (interferon-stimulated gene 15) regulates the host response to bacterial and viral infections through its conjugation proteins (ISGylation) following interferon production. ISGylation is antagonized by highly specific cysteine protease USP18, which major deISGylating enzyme. However, mechanisms underlying USP18's extraordinary specificity towards remains elusive. Here, we show that USP18 interacts with paralog USP41, whose catalytic domain shares 97% identity USP18. USP41 does not act as a deISGylase, led us perform comparative analysis decipher basis for this difference, revealing molecular determinants of ISG15. We found C-terminus, well conserved Leucine at position 198, are essential enzymatic activity likely functional surfaces based on AlphaFold predictions. Finally, propose antagonizes understudied FAT10 (HLA-F adjacent transcript 10) from substrates in catalytic-independent manner. Altogether, our results offer new insights into ISG15, while identifying negative regulator conjugation.

Language: Английский

Citations

0
USP24 is an ISG15 cross-reactive deubiquitinase that mediates IFN-I production by de-ISGylating the RNA helicase MOV10 DOI Creative Commons
Rishov Mukhopadhyay, Simeon D. Draganov, Jimmy J.L.L. Akkermans

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 6, 2024

ABSTRACT The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays crucial role in the innate immune response against viral infections. ISG15 conjugated to target proteins an enzymatic cascade through process called ISGylation. While ubiquitin-specific protease 18 (USP18) well-defined deISGylase counteracting conjugation, cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as novel DUB identified activity-based protein profiling (ABPP). We demonstrate that recombinant processed pro-ISG15 ISG15-linked synthetic substrates vitro . Moreover, depletion of significantly increased accumulation conjugates upon IFN-β stimulation. An extensive proteomic analysis USP24-dependent ISGylome, integrating total proteome, GG-peptidome, interactome data, helicase Moloney leukemia virus 10 (MOV10) specific for deISGylation. Further validation cells revealed ISGylated MOV10 enhances production/secretion, whereas deISGylates negatively regulate response. This showcases USP24’s roles modulating ISGylation modulation IFN-I-dependent responses, with potential therapeutic implications infectious diseases, cancer, autoimmunity, neuroinflammation.

Language: Английский

Citations

0
A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals DOI Creative Commons

Anahi G. Jimenez-Campos,

Lucas I. Maestas,

Nileena Velappan

et al.

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0309305 - e0309305

Published: Dec. 26, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple fast tests that reliably detect active infections with SARS-CoV-2 in the early stage of infection. Here we introduce rapid activity-based diagnostic (ABDx) test identifies by measuring activity viral enzyme, Papain-Like protease (PLpro). The system consists peptide fluoresces when cleaved SARS PLpro is crude, unprocessed lysates from tongue scrapes saliva. Test results obtained 30 minutes or less using widely available fluorescence plate readers, battery-operated portable instrument on-site testing. Proof-of-concept was study on clinical specimens collected patients COVID-19 like symptoms who tested positive (n = 10) negative LIAT RT-PCR nasal mid turbinate swabs. When saliva these in-house endpoint RT-PCR, 17 were only 5 negative, which became days later. correlated cases (3 out 3 negatives 14 16 positives, one invalid specimen). Despite small number samples, agreement significant (p value 0.01). Two false detected, sample late Ct 35 indicating an infection no longer present. assay easily scalable expected all viable variants, making it attractive as screening surveillance tool. Additionally, show feasibility platform new homogeneous phenotypic antiviral drugs neutralizing antibodies.

Language: Английский

Citations

0