Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611129
Laísa Quadros Barsé, Candida Deves Roth, Adilio da Silva Dadda, Raoní Scheibler Rambo, Pedro Ferrari Dalberto, Kenia Pissinate, José Eduardo Sacconi Nunes, Renata Jardim Etchart, Pablo Machado, Luiz A Basso, Cristiano Valim Bizarro
Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis (Mtb) and is responsible for millions of deaths. New Mtb strains resistant to TB drugs are emerging and spreading. The first-line TB drug, isoniazid (INH), must be activated inside mycobacterial cells by the catalase-peroxidase enzyme KatG to exert its antimicrobial activity, and mutations on the katG gene are a significant cause of INH resistance in clinics. The metal-containing compound IQG-607 is an INH analog developed to inhibit the target of INH, the FASII enzyme enoyl-ACP-reductase (InhA), without requiring KatG. However, we recently showed that inside mycobacterial cells, IQG-607 activity depends on KatG. Hence, this compound might also be activated by KatG to inhibit InhA. We evaluated whether recombinant MtKatG uses IQG-607 as a substrate in oxidation reactions and adduct formation with NAD+. A recombinant MtKatG was produced in E. coli and purified in a 3-step protocol to obtain a homogeneous protein. An HPLC method was optimized to monitor both oxidation and adduct products, and our assay system was validated by performing control reactions using INH as a substrate. We found that the metal-based compound IQG-607 is not a substrate for recombinant MtKatG under all conditions tested.
{"title":"The isoniazid analog IQG-607 is not a direct substrate for the Mycobacterium tuberculosis catalase-peroxidase KatG","authors":"Laísa Quadros Barsé, Candida Deves Roth, Adilio da Silva Dadda, Raoní Scheibler Rambo, Pedro Ferrari Dalberto, Kenia Pissinate, José Eduardo Sacconi Nunes, Renata Jardim Etchart, Pablo Machado, Luiz A Basso, Cristiano Valim Bizarro","doi":"10.1101/2024.09.05.611129","DOIUrl":"https://doi.org/10.1101/2024.09.05.611129","url":null,"abstract":"Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis (Mtb) and is responsible for millions of deaths. New Mtb strains resistant to TB drugs are emerging and spreading. The first-line TB drug, isoniazid (INH), must be activated inside mycobacterial cells by the catalase-peroxidase enzyme KatG to exert its antimicrobial activity, and mutations on the katG gene are a significant cause of INH resistance in clinics. The metal-containing compound IQG-607 is an INH analog developed to inhibit the target of INH, the FASII enzyme enoyl-ACP-reductase (InhA), without requiring KatG. However, we recently showed that inside mycobacterial cells, IQG-607 activity depends on KatG. Hence, this compound might also be activated by KatG to inhibit InhA. We evaluated whether recombinant MtKatG uses IQG-607 as a substrate in oxidation reactions and adduct formation with NAD+. A recombinant MtKatG was produced in E. coli and purified in a 3-step protocol to obtain a homogeneous protein. An HPLC method was optimized to monitor both oxidation and adduct products, and our assay system was validated by performing control reactions using INH as a substrate. We found that the metal-based compound IQG-607 is not a substrate for recombinant MtKatG under all conditions tested.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"2016 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.06.611642
Justyne L Ogdahl, Peter Chien
The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.
ATPase Associated with diverse cellular Activities（AAA+）蛋白酶家族在细胞蛋白分解和应激反应中发挥着至关重要的作用。与其他 AAA+ 蛋白酶一样，已知 Lon 蛋白酶受核苷酸和底物结合的异构调节。虽然它最初被归类为 DNA 结合蛋白，但 DNA 结合对 Lon 活性的影响尚不清楚。在这项研究中，我们描述了单链 DNA（ssDNA）结合对 Lon 的调控，并偶然发现了 Lon 的一般激活策略。与 ssDNA 结合后，Lon 的 ATP 水解速率会因核苷酸结合的改善而增加，从而导致蛋白质底物（包括重要的生理靶标）的降解增强。我们证明，对 Lon 的 DNA 结合至关重要的基本残基发生突变，不仅会减少 ssDNA 的结合，还会对 Lon 的活性产生电荷特异性影响。在这些位点引入负电荷会诱导类似于 ssDNA 结合所诱导的活化，而中和电荷则会降低 Lon 的活性。基于单分子测量，我们发现这种活性变化与 Lon 寡聚化的变化相关。我们的研究深入揭示了 DNA 结合或突变所产生的静电作用对 Lon 蛋白酶的复杂调控。
{"title":"Allosteric modulation of the Lon protease by effector binding and local charges","authors":"Justyne L Ogdahl, Peter Chien","doi":"10.1101/2024.09.06.611642","DOIUrl":"https://doi.org/10.1101/2024.09.06.611642","url":null,"abstract":"The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"63 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611390
Rebecca Roddan, William J. Nathan, Joseph A. Newman, Afaf H. El-Sagheer, David M. Wilson, Tom Brown, Christopher J. Schofield, Peter J. McHugh
The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro, implying a key role of the interaction in ′unhooking′ during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB′s stimulation of SNM1A′s activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB′s stimulation of SNM1A′s nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.
SNM1A 外切酶在链间交联（ICL）的修复中发挥着关键作用，链间交联是一类毒性特别强的 DNA 损伤。以前的研究表明，在转录耦合 DNA 链间交联修复（TC-ICL 修复）过程中，SWI/SNF 家族 ATP 依赖性染色质重塑因子 Cockayne 综合征 B 蛋白（CSB）与 SNM1A 相互作用。在这里，我们利用纯化的蛋白质验证了这种相互作用，并证明 CSB 的泛素结合结构域和翼螺旋结构域是与 SNM1A 催化结构域相互作用所必需的。翼螺旋结构域是结合的必要条件，但高亲和性 SNM1A 结合需要整个 CSB C 端区域（残基 1187-1493），其中 CSB C 端结构域的两个拷贝是与 SNM1A 稳定相互作用的必要条件。CSB 能刺激 SNM1A 在各种 DNA 修复中间底物模型上的核酸酶活性。重要的是，在体外观察到 CSB 能刺激 ICL 的消化，这意味着在 TC-ICL 修复过程中，CSB 与 SNM1A 的相互作用在 "解钩 "过程中起着关键作用。与 SNM1A 催化结构域复合的 CSB 构建物的 AlphaFold3 模型能够绘制 CSB-SNM1A 相互作用所需的分子接触图。这确定了 CSB 刺激 SNM1A 活性所必需的特定蛋白间相互作用，我们通过实验证实了这一点。此外，我们的研究还发现 CSB 的 C 端区域是一个新的 DNA 结合区域，它也参与刺激 SNM1A 介导的 ICL 修复。此外，靶向对特定核酸酶活性至关重要的蛋白-蛋白相互作用，如 CSB 对 SNM1A 核酸酶活性的刺激，可能是核酸酶活性位点抑制之外的另一种有效治疗策略。
{"title":"Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing","authors":"Rebecca Roddan, William J. Nathan, Joseph A. Newman, Afaf H. El-Sagheer, David M. Wilson, Tom Brown, Christopher J. Schofield, Peter J. McHugh","doi":"10.1101/2024.09.05.611390","DOIUrl":"https://doi.org/10.1101/2024.09.05.611390","url":null,"abstract":"The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro, implying a key role of the interaction in ′unhooking′ during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB′s stimulation of SNM1A′s activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB′s stimulation of SNM1A′s nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611238
Adam K Hedger, Wazo Myint, Jeong Min Lee, Diego Suchenski-Loustaunau, Vanivilasini Balachandran, Ala M Shaqra, Nese Kurt Yilmaz, Jonathan Watts, Hiroshi Matsuo, Celia A Schiffer
APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.
{"title":"Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability","authors":"Adam K Hedger, Wazo Myint, Jeong Min Lee, Diego Suchenski-Loustaunau, Vanivilasini Balachandran, Ala M Shaqra, Nese Kurt Yilmaz, Jonathan Watts, Hiroshi Matsuo, Celia A Schiffer","doi":"10.1101/2024.09.05.611238","DOIUrl":"https://doi.org/10.1101/2024.09.05.611238","url":null,"abstract":"APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.06.611391
Rishov Mukhopadhyay, Simeon D Draganov, Jimmy J.L.L Akkermans, Marjolein Kikkert, Klaus-Peter Knobeloch, Günter Fritz, María Guzmán, Sonia Zuñiga, Robbert Q Kim, Benedikt M Kessler, Adán Pinto-Fernández, Paul P Geurink, Aysegul Sapmaz
The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-β stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-β production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24's novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.
{"title":"USP24 is an ISG15 cross-reactive deubiquitinase that mediates IFN-I production by de-ISGylating the RNA helicase MOV10","authors":"Rishov Mukhopadhyay, Simeon D Draganov, Jimmy J.L.L Akkermans, Marjolein Kikkert, Klaus-Peter Knobeloch, Günter Fritz, María Guzmán, Sonia Zuñiga, Robbert Q Kim, Benedikt M Kessler, Adán Pinto-Fernández, Paul P Geurink, Aysegul Sapmaz","doi":"10.1101/2024.09.06.611391","DOIUrl":"https://doi.org/10.1101/2024.09.06.611391","url":null,"abstract":"The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-β stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-β production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24's novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1101/2024.09.05.611520
Erin Skeens, Federica Maschietto, Manjula Ramu, Shanelle Shillingford, Elias J Lolis, Victor S Batista, Anton M Bennett, George P Lisi
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.
{"title":"Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase","authors":"Erin Skeens, Federica Maschietto, Manjula Ramu, Shanelle Shillingford, Elias J Lolis, Victor S Batista, Anton M Bennett, George P Lisi","doi":"10.1101/2024.09.05.611520","DOIUrl":"https://doi.org/10.1101/2024.09.05.611520","url":null,"abstract":"Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1101/2024.09.05.611540
Giang T Nguyen, Michael A Schelling, Dipali G Sashital
Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.
Cas 内切酶，如 Cas9 和 Cas12a，是一种 RNA 引导的免疫效应器，可帮助细菌抵御噬菌体。Cas 内切酶依靠二价金属离子进行酶促活动，并促进构象变化，而构象变化是特异性识别和切割目标 DNA 所必需的。虽然 Cas 内切酶通常会在 DNA 靶标上产生双链断裂（DSB），但生理相关的 Mg2+ 浓度降低和靶标错配会导致不完全的第二链裂解，从而产生缺口 DNA。人们对 Cas 内切酶的切口是否足以提供对噬菌体的保护仍知之甚少。为了解决这个问题，我们测试了Cas9缺口酶对噬菌体的保护作用，在Cas9缺口酶中，两个核酸酶结构域中只有一个具有催化活性。通过测试大量的引导 RNA，我们发现靶向链切分足以提供免疫力，而非靶向切分除了与 Cas9 结合外并不能提供额外的保护。靶向链切割能抑制噬菌体的复制，当靶向基因组的非必要区域时，能降低 Cas9 被病毒逃脱的可能性。在 Mg2+ 浓度较低时，RuvC 结构域对非目标链的切割会受到严重影响。因此，能与游离 Mg2+ 竞争结合的其他生物大分子浓度的波动会强烈影响 Cas9 在靶位点形成 DSB 的能力。总之，我们的研究结果表明，在CRISPR介导的免疫过程中，Cas9只能对DNA进行切口，尤其是在细胞中Mg2+含量较低的条件下。
{"title":"CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication","authors":"Giang T Nguyen, Michael A Schelling, Dipali G Sashital","doi":"10.1101/2024.09.05.611540","DOIUrl":"https://doi.org/10.1101/2024.09.05.611540","url":null,"abstract":"Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg<sup>2+</sup> concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg<sup>2+</sup> strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"116 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1101/2024.09.05.611400
Anna Pagotto, Federico Uliana, Giulia Nordio, Andrea Pietrangelini, Laura Acquasaliente, Maria Ludovica Macchia, Massimo Bellanda, Barbara Gatto, Giustina De Silvestro, Piero Marson, Paolo Simioni, Paola Picotti, Vincenzo De Filippis
Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.
{"title":"The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII","authors":"Anna Pagotto, Federico Uliana, Giulia Nordio, Andrea Pietrangelini, Laura Acquasaliente, Maria Ludovica Macchia, Massimo Bellanda, Barbara Gatto, Giustina De Silvestro, Piero Marson, Paolo Simioni, Paola Picotti, Vincenzo De Filippis","doi":"10.1101/2024.09.05.611400","DOIUrl":"https://doi.org/10.1101/2024.09.05.611400","url":null,"abstract":"Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1101/2024.09.05.611393
Julia Schoenfeld, Steffen Brunst, Ludmila Ciomirtan, Nick Liebisch, Adarsh Kumar, Johanna Ehrler, Lukas Wintermeier, Jan Heering, Astrid Brueggerhof, Lilia Weizel, Astrid Kahnt, Manfred Schubert-Zsilavecz, Stefan Knapp, Robert Fuerst, Eugen Proschak, Kerstin Hiesinger
Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker. Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimer's disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.
{"title":"STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE","authors":"Julia Schoenfeld, Steffen Brunst, Ludmila Ciomirtan, Nick Liebisch, Adarsh Kumar, Johanna Ehrler, Lukas Wintermeier, Jan Heering, Astrid Brueggerhof, Lilia Weizel, Astrid Kahnt, Manfred Schubert-Zsilavecz, Stefan Knapp, Robert Fuerst, Eugen Proschak, Kerstin Hiesinger","doi":"10.1101/2024.09.05.611393","DOIUrl":"https://doi.org/10.1101/2024.09.05.611393","url":null,"abstract":"Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker. Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimer's disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1101/2024.09.05.611490
Assmaa Elsheikh, Camden M Driggers, Ha H. Truong, Zhongying Yang, John Allen, Niel Henriksen, Katarzyna Walczewska-Szewc, Show-Ling Shyng
Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ~ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.
{"title":"AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone","authors":"Assmaa Elsheikh, Camden M Driggers, Ha H. Truong, Zhongying Yang, John Allen, Niel Henriksen, Katarzyna Walczewska-Szewc, Show-Ling Shyng","doi":"10.1101/2024.09.05.611490","DOIUrl":"https://doi.org/10.1101/2024.09.05.611490","url":null,"abstract":"Pancreatic K<sub>ATP</sub> channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the K<sub>ATP</sub> channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used K<sub>ATP</sub> channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for K<sub>ATP</sub> trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of K<sub>ATP</sub> channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known K<sub>ATP</sub> pharmacochaperones bind. The structural knowledge provides a framework for discovering K<sub>ATP</sub> channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on K<sub>ATP</sub> channel trafficking mutations. Aekatperone reversibly inhibits K<sub>ATP</sub> channel activity with a half-maximal inhibitory concentration (IC50) ~ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of K<sub>ATP</sub> bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a K<sub>ATP</sub> pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by K<sub>ATP</sub> trafficking defects.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"60 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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