scholarly journals Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role in Betacoronavirus replication

2021 ◽  
Vol 118 (49) ◽  
pp. e2108709118
Author(s):  
Natacha S. Ogando ◽  
Priscila El Kazzi ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Brenda W. Bontes ◽  
Alice Decombe ◽  
...  
Keyword(s):  
Function Analysis ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Messenger Rnas ◽  
Mtase Activity ◽  
Mrna Capping ◽  
Activity Spectrum ◽  
Immune Sensing ◽  
Key Residues

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5′ exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal (N7-guanine)–methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)–CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

scholarly journals Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in betacoronavirus replication

2021 ◽  
Author(s):  
Natacha S. Ogando ◽  
Priscila El Kazzi ◽  
Clara C S. Posthuma ◽  
Volker Thiel ◽  
Bruno Canard ◽  
...  
Keyword(s):  
Function Analysis ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Viral Progeny ◽  
Mrna Capping ◽  
Activity Spectrum ◽  
Immune Sensing ◽  
Key Residues ◽  
Innate Immune Sensing

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3-to-5exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is assumed to be involved in viral mRNA capping. Here, we first revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between different betacoronaviruses (beta-CoVs). In this study, we identified several residues likely to be involved in the formation of the catalytic pocket of N7MTase, which presents a fold that is distinct from the Rossmann fold observed in most known MTases. Next, for multiple beta-CoVs, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity and viral replication in cell culture. For SARS-CoV and Middle East respiratory syndrome-CoV, most of the engineered mutations abolished the N7-MTase function, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into beta-CoV genomes, we identified two substitutions (R310A and F426A in SARS-CoV) that abrogated viral progeny production and one mutation (H424A) that yielded a crippled phenotype across all beta-CoVs tested. Our results identify the N7-MTase as a critical enzyme for beta-CoV replication and defined key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

scholarly journals Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation

2020 ◽  
Vol 117 (3) ◽  
pp. 1429-1437 ◽  
Author(s):  
Bridget Y. Huang ◽  
Israel S. Fernández
Keyword(s):  
Translation Initiation ◽  
Site Directed Mutagenesis ◽  
Messenger Rnas ◽  
Gtp Hydrolysis ◽  
Dead End ◽  
Initiation Reaction ◽  
Set Up ◽  
High Resolution Reconstruction

Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a “dead-end” intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.

scholarly journals Structure/Function Analysis of Neisseria meningitidis PilW, a Conserved Protein That Plays Multiple Roles in Type IV Pilus Biology

2011 ◽  
Vol 79 (8) ◽  
pp. 3028-3035 ◽  
Author(s):  
Tim H. Szeto ◽  
Andréa Dessen ◽  
Vladimir Pelicic
Keyword(s):  
Structure Function ◽  
Neisseria Meningitidis ◽  
Function Analysis ◽  
Site Directed Mutagenesis ◽  
Human Pathogens ◽  
Content Type ◽  
Multifunctional Protein ◽  
Bacterial Surface ◽  
Type Iv ◽  
Key Residues

ABSTRACTType IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple functions and play a key role in pathogenesis in several important human pathogens, includingNeisseria meningitidis. Tfp biology remains poorly understood at a molecular level because the roles of the numerous proteins that are involved remain mostly obscure. Guided by the high-resolution crystal structure we recently reported forN. meningitidisPilW, a widely conserved protein essential for Tfp biogenesis, we have performed a structure/function analysis by targeting a series of key residues through site-directed mutagenesis and analyzing the corresponding variants using an array of phenotypic assays. Here we show that PilW's involvement in the functionality of Tfp can be genetically uncoupled from its concurrent role in the assembly/stabilization of the secretin channels through which Tfp emerge on the bacterial surface. These findings suggest that PilW is a multifunctional protein.

scholarly journals Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors

2021 ◽  
pp. 247255522110262
Author(s):  
Kanchan Devkota ◽  
Matthieu Schapira ◽  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Fengling Li ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Competitive Inhibitor ◽  
Human Immune System ◽  
Mtase Activity ◽  
Competitive Inhibitors ◽  
Human Immune ◽  
Rna Capping ◽  
Socioeconomic Consequences ◽  
Bisubstrate Inhibitors

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure–activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

scholarly journals Identification of Natural Molecular Determinants of Ross River Virus Type I Interferon Modulation

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Xiang Liu ◽  
Margit Mutso ◽  
Liubov Cherkashchenko ◽  
Eva Zusinaite ◽  
Lara J. Herrero ◽  
...  
Keyword(s):  
Amino Acid ◽  
Type I Interferon ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Type I Interferons ◽  
Ross River Virus ◽  
Type I ◽  
Type I Ifn ◽  
Nonstructural Protein 1

ABSTRACT Ross River virus (RRV) belongs to the genus Alphavirus and is prevalent in Australia. RRV infection can cause arthritic symptoms in patients and may include rash, fever, arthralgia, and myalgia. Type I interferons (IFN) are the primary antiviral cytokines and trigger activation of the host innate immune system to suppress the replication of invading viruses. Alphaviruses are able to subvert the type I IFN system, but the mechanisms used are ill defined. In this study, seven RRV field strains were analyzed for induction of and sensitivity to type I IFN. The sensitivities of these strains to human IFN-β varied significantly and were highest for the RRV 2548 strain. Compared to prototype laboratory strain RRV-T48, RRV 2548 also induced higher type I IFN levels both in vitro and in vivo and caused milder disease. To identify the determinants involved in type I IFN modulation, the region encoding the nonstructural proteins (nsPs) of RRV 2548 was sequenced, and 42 amino acid differences from RRV-T48 were identified. Using fragment swapping and site-directed mutagenesis, we discovered that substitutions E402A and R522Q in nsP1 as well as Q619R in nsP2 were responsible for increased sensitivity of RRV 2548 to type I IFN. In contrast, substitutions A31T, N219T, S580L, and Q619R in nsP2 led to induction of higher levels of type I IFN. With exception of E402A, all these variations are common for naturally occurring RRV strains. However, they are different from all known determinants of type I IFN modulation reported previously in nsPs of alphaviruses. IMPORTANCE By identifying natural Ross River virus (RRV) amino acid determinants for type I interferon (IFN) modulation, this study gives further insight into the mechanism of type I IFN modulation by alphaviruses. Here, the crucial role of type I IFN in the early stages of RRV disease pathogenesis is further demonstrated. This study also provides a comparison of the roles of different parts of the RRV nonstructural region in type I IFN modulation, highlighting the importance of nonstructural protein 1 (nsP1) and nsP2 in this process. Three substitutions in nsP1 and nsP2 were found to be independently associated with enhanced type I IFN sensitivity, and four independent substitutions in nsP2 were important in elevated type I IFN induction. Such evidence has clear implications for RRV immunobiology, persistence, and pathology. The identification of viral proteins that modulate type I IFN may also have importance for the pathogenesis of other alphaviruses.

scholarly journals Mutagenesis ofS-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity

2016 ◽  
Vol 90 (16) ◽  
pp. 7248-7256 ◽  
Author(s):  
James Brett Case ◽  
Alison W. Ashbrook ◽  
Terence S. Dermody ◽  
Mark R. Denison
Keyword(s):  
Viral Replication ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Rna Stability ◽  
Type I ◽  
Translation Efficiency ◽  
Murine Hepatitis Virus ◽  
Mtase Activity ◽  
Binding Residues

ABSTRACTEukaryotic mRNAs possess a methylated 5′-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5′ caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conservedS-adenosyl-l-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activityin vitro. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently bothin vitroand during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differin vitroand during virus infection.IMPORTANCEHuman coronaviruses, most notably severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that substitution of alanine at G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.

scholarly journals mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

2015 ◽  
Vol 89 (16) ◽  
pp. 8292-8303 ◽  
Author(s):  
Changqing Li ◽  
Jaime Guillén ◽  
Nadia Rabah ◽  
Alexandre Blanjoie ◽  
Françoise Debart ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Health Concern ◽  
Viral Mrna ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Mrna Capping ◽  
First Time

ABSTRACTAlphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group fromS-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m7GMP-nsP1 adduct. Subsequent transfer of m7GMP onto the 5′ end of the viral mRNA has not been demonstratedin vitroyet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m7G-cap monoclonal antibody, respectively. The GT reaction is stimulated byS-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m7GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5′-diphosphate RNA oligonucleotide whose sequence corresponds to the 5′ end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.IMPORTANCEEmergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5′ diphosphate synthetic RNA mimicking the 5′ end of an alphavirus mRNA was observedin vitrofor the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.

scholarly journals Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas

2013 ◽  
Vol 42 (5) ◽  
pp. 3286-3297 ◽  
Author(s):  
Karen Loizeau ◽  
Yujiao Qu ◽  
Sébastien Depp ◽  
Vincent Fiechter ◽  
Hannes Ruwe ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Chloroplast Transformation ◽  
Site Directed Mutagenesis ◽  
Tetratricopeptide Repeat ◽  
Messenger Rnas ◽  
Mrna Accumulation ◽  
Cis Acting

Abstract Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5′-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5′ UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5′ UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5′ UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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