Pub Date : 2024-04-29DOI: 10.1021/acs.biochem.4c00049
Samuel Cashman-Kadri, Patrick Lagüe, Muriel Subirade, Ismail Fliss and Lucie Beaulieu*,
Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.
{"title":"Insights into Molecular Interactions between a GAPDH-Related Fish Antimicrobial Peptide, Analogs Thereof, and Bacterial Membranes","authors":"Samuel Cashman-Kadri, Patrick Lagüe, Muriel Subirade, Ismail Fliss and Lucie Beaulieu*, ","doi":"10.1021/acs.biochem.4c00049","DOIUrl":"10.1021/acs.biochem.4c00049","url":null,"abstract":"<p >Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00049","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140835549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-28DOI: 10.1021/acs.biochem.4c00028
Collin J. Steen*, Jens Niklas, Oleg G. Poluektov, Richard D. Schaller, Graham R. Fleming and Lisa M. Utschig,
A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).
{"title":"EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis","authors":"Collin J. Steen*, Jens Niklas, Oleg G. Poluektov, Richard D. Schaller, Graham R. Fleming and Lisa M. Utschig, ","doi":"10.1021/acs.biochem.4c00028","DOIUrl":"10.1021/acs.biochem.4c00028","url":null,"abstract":"<p >A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (<sup>1</sup>O<sub>2</sub>*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of <sup>1</sup>O<sub>2</sub>* in photodamage and cell signaling, few studies directly correlate <sup>1</sup>O<sub>2</sub>* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of <sup>1</sup>O<sub>2</sub>* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of <sup>1</sup>O<sub>2</sub>* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of <sup>1</sup>O<sub>2</sub>* in chlorophyll-containing photosynthetic membranes. We find that the apparent <sup>1</sup>O<sub>2</sub>* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of <sup>1</sup>O<sub>2</sub>* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of <sup>1</sup>O<sub>2</sub>* during high light. Upon saturation of NPQ, the concentration of <sup>1</sup>O<sub>2</sub>* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00028","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140835830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26DOI: 10.1021/acs.biochem.3c00670
Corey Thomas, Lisbeth Avalos-Irving, Jorge Victorino, Sydney Green, Morgan Andrews, Naisha Rodrigues, Sarah Ebirim, Ayden Mudd and Jamie B. Towle-Weicksel*,
DNA polymerase θ (Pol θ or POLQ) is primarily involved in repairing double-stranded breaks in DNA through an alternative pathway known as microhomology-mediated end joining (MMEJ) or theta-mediated end joining (TMEJ). Unlike other DNA repair polymerases, Pol θ is thought to be highly error-prone yet critical for cell survival. We have identified several POLQ gene variants from human melanoma tumors that experience altered DNA polymerase activity, including a propensity for incorrect nucleotide selection and reduced polymerization rates compared to WT Pol θ. Variants are 30-fold less efficient at incorporating a nucleotide during repair and up to 70-fold less accurate at selecting the correct nucleotide opposite a templating base. This suggests that aberrant Pol θ has reduced DNA repair capabilities and may also contribute to increased mutagenesis. Moreover, the variants were identified in established tumors, suggesting that cancer cells may use mutated polymerases to promote metastasis and drug resistance.
DNA 聚合酶θ(Pol θ 或 POLQ)主要通过一种被称为微同源物介导的末端连接(MMEJ)或θ介导的末端连接(TMEJ)的替代途径参与 DNA 双链断裂的修复。与其他 DNA 修复聚合酶不同,Pol θ 被认为极易出错,但对细胞存活至关重要。我们从人类黑色素瘤肿瘤中发现了几种 POLQ 基因变异体,它们的 DNA 聚合酶活性发生了改变,包括核苷酸选择错误的倾向以及与 WT Pol θ 相比聚合率降低。变异体在修复过程中整合核苷酸的效率要低 30 倍,选择与模板碱基相对的正确核苷酸的准确性也要低 70 倍。这表明,畸变 Pol θ 的 DNA 修复能力降低,也可能导致突变增加。此外,这些变体是在已确诊的肿瘤中发现的,这表明癌细胞可能利用突变的聚合酶来促进转移和耐药性。
{"title":"Melanoma-Derived DNA Polymerase Theta Variants Exhibit Altered DNA Polymerase Activity","authors":"Corey Thomas, Lisbeth Avalos-Irving, Jorge Victorino, Sydney Green, Morgan Andrews, Naisha Rodrigues, Sarah Ebirim, Ayden Mudd and Jamie B. Towle-Weicksel*, ","doi":"10.1021/acs.biochem.3c00670","DOIUrl":"10.1021/acs.biochem.3c00670","url":null,"abstract":"<p >DNA polymerase θ (Pol θ or POLQ) is primarily involved in repairing double-stranded breaks in DNA through an alternative pathway known as microhomology-mediated end joining (MMEJ) or theta-mediated end joining (TMEJ). Unlike other DNA repair polymerases, Pol θ is thought to be highly error-prone yet critical for cell survival. We have identified several POLQ gene variants from human melanoma tumors that experience altered DNA polymerase activity, including a propensity for incorrect nucleotide selection and reduced polymerization rates compared to WT Pol θ. Variants are 30-fold less efficient at incorporating a nucleotide during repair and up to 70-fold less accurate at selecting the correct nucleotide opposite a templating base. This suggests that aberrant Pol θ has reduced DNA repair capabilities and may also contribute to increased mutagenesis. Moreover, the variants were identified in established tumors, suggesting that cancer cells may use mutated polymerases to promote metastasis and drug resistance.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.3c00670","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140804271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26DOI: 10.1021/acs.biochem.3c00668
Tania Churasacari Vinces, Anacleto Silva de Souza, Cecília F. Carvalho, Aline Biazola Visnardi, Raphael D. Teixeira, Edgar E. Llontop, Beatriz Aparecida Passos Bismara, Elisabete J. Vicente, José O. Pereira, Robson Francisco de Souza, Mauricio Yonamine, Sandro Roberto Marana, Chuck Shaker Farah and Cristiane R. Guzzo*,
Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.
{"title":"Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy","authors":"Tania Churasacari Vinces, Anacleto Silva de Souza, Cecília F. Carvalho, Aline Biazola Visnardi, Raphael D. Teixeira, Edgar E. Llontop, Beatriz Aparecida Passos Bismara, Elisabete J. Vicente, José O. Pereira, Robson Francisco de Souza, Mauricio Yonamine, Sandro Roberto Marana, Chuck Shaker Farah and Cristiane R. Guzzo*, ","doi":"10.1021/acs.biochem.3c00668","DOIUrl":"10.1021/acs.biochem.3c00668","url":null,"abstract":"<p >Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as <i>p</i>-nitrophenyl butyrate and <i>p</i>-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅<sub>1</sub>). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅<sub>2</sub>). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140804234","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26DOI: 10.1021/acs.biochem.3c00624
Shahid A. Malik*, Somnath Mondal* and Hanudatta S. Atreya,
Parkinson’s disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein’s inclination toward quicker aggregation is explained by the binding of the N-terminus of α-syn with the gold nanoparticles. Conversely, a comparatively stronger interaction with GO causes the nucleation and growth phases to be postponed and inhibits intermolecular interactions. Our finding offers novel experimental insights at the residue level regarding the aggregation of α-syn in the presence of various nanomaterials and creates new opportunities for the development of suitably functionalized nanomaterial-based therapeutic reagents against Parkinson’s and other neurodegenerative diseases.
帕金森病(PD)的特点是单体α-突触核蛋白(α-syn)形成有毒的低聚物和纤维相。某些纳米粒子已被证实能促进蛋白质的聚集,而其他纳米材料则能阻止这一过程。在目前的工作中,我们使用核磁共振光谱法结合等温滴定量热法,在氨基酸蛋白质水平上研究这些相反效应的原因和机制。我们考虑了 α-syn 与两种纳米材料的相互作用:柠檬酸盐帽金纳米粒子(AuNPs)和氧化石墨烯(GO)。在 AuNPs 的存在下,α-syn 会加速聚集,而在 GO 的存在下,α-syn 的聚集会被阻止。研究表明,GO 通过静电和疏水相互作用封存了 α-syn 单体的 NAC 区域,导致伸长率降低,而 AuNPs 则在结合 N 端的同时让 NAC 区域暴露在外,导致更高的聚集率。α-syn的N端与金纳米粒子的结合解释了蛋白质倾向于更快聚集的原因。相反,与 GO 相对较强的相互作用会推迟成核和生长阶段,并抑制分子间的相互作用。我们的发现为α-syn在各种纳米材料存在下的聚集提供了残基水平上的新实验见解,并为开发基于纳米材料的帕金森病和其他神经退行性疾病的适当功能化治疗试剂创造了新的机会。
{"title":"Alpha-Synuclein Aggregation Mechanism in the Presence of Nanomaterials","authors":"Shahid A. Malik*, Somnath Mondal* and Hanudatta S. Atreya, ","doi":"10.1021/acs.biochem.3c00624","DOIUrl":"10.1021/acs.biochem.3c00624","url":null,"abstract":"<p >Parkinson’s disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein’s inclination toward quicker aggregation is explained by the binding of the N-terminus of α-syn with the gold nanoparticles. Conversely, a comparatively stronger interaction with GO causes the nucleation and growth phases to be postponed and inhibits intermolecular interactions. Our finding offers novel experimental insights at the residue level regarding the aggregation of α-syn in the presence of various nanomaterials and creates new opportunities for the development of suitably functionalized nanomaterial-based therapeutic reagents against Parkinson’s and other neurodegenerative diseases.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140651966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26DOI: 10.1021/acs.biochem.4c00117
Angela M. Develin, and , Brian Fuglestad*,
As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47phox translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47phox recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47phox-PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47phox membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47phox is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.
{"title":"Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47phox Membrane Anchoring","authors":"Angela M. Develin, and , Brian Fuglestad*, ","doi":"10.1021/acs.biochem.4c00117","DOIUrl":"10.1021/acs.biochem.4c00117","url":null,"abstract":"<p >As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47<sup>phox</sup> translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47<sup>phox</sup> recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47<sup>phox</sup>-PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47<sup>phox</sup> membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47<sup>phox</sup> is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00117","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140804220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-25DOI: 10.1021/acs.biochem.4c00046
Diana L. Cudia, Effibe O. Ahoulou, Aritra Bej, Annika N. Janssen, Alexander Scholten, Karl-W. Koch and James B. Ames*,
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
{"title":"NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation","authors":"Diana L. Cudia, Effibe O. Ahoulou, Aritra Bej, Annika N. Janssen, Alexander Scholten, Karl-W. Koch and James B. Ames*, ","doi":"10.1021/acs.biochem.4c00046","DOIUrl":"10.1021/acs.biochem.4c00046","url":null,"abstract":"<p >Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5<sup>R22A</sup>) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5<sup>WT</sup>). Here, we present ITC, NMR, and functional analysis of GCAP5<sup>R22A</sup> to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5<sup>R22A</sup> binds a total of 3 Ca<sup>2+</sup>, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5<sup>WT</sup> were not detected by ITC, suggesting that R22A may affect the binding of Ca<sup>2+</sup> to these sites. The NMR-derived structure of GCAP5<sup>R22A</sup> is overall similar to that of GCAP5<sup>WT</sup> (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5<sup>R22A</sup> lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5<sup>R22A</sup> with ∼25% higher affinity compared to GCAP5<sup>WT</sup>, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140653439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-19DOI: 10.1021/acs.biochem.4c00132
Charu Thapliyal*, and , Rajesh Mishra*,
HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB’s intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.
{"title":"The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity","authors":"Charu Thapliyal*, and , Rajesh Mishra*, ","doi":"10.1021/acs.biochem.4c00132","DOIUrl":"10.1021/acs.biochem.4c00132","url":null,"abstract":"<p >HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB’s intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140682559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-16DOI: 10.1021/acs.biochem.3c00646
Megh R. Bhatt, Himal K. Ganguly and Neal J. Zondlo*,
Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4–N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X–AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water–amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide–water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.
相对于游离的 N 端,酰基封端基团通过提供一个额外的 C═Oi---Hi+4-N 氢键来稳定 α 螺旋。酰基封端基团的电子特性也可能直接调节α-螺旋的稳定性:富含电子的 N 端酰基基团可以通过加强 i/i + 4 氢键和 i/i + 1 n → π* 相互作用来稳定α-螺旋。这一假设在肽 X-AKAAAAKAAAAKAAGY-NH2(其中 X = 不同的酰基)中得到了验证。令人惊讶的是,电子最丰富的酰基(新戊酰基和异丁酰基)强烈地破坏了α-螺旋的稳定性。此外,尽管甲酰基在氢键和 n → π* 相互作用中是较弱的电子供体,但它诱导的 α 螺旋度几乎与乙酰基相同。其他酰基则表现出中等的α-螺旋性。这些结果表明,酰基羰基的电子特性并不能直接决定肽在水中的α-helicity。为了了解这些影响,我们对 α 螺旋肽进行了 DFT 计算。利用隐式溶解,α-螺旋稳定性与酰基电子相关,与实验结果相反,新戊酰基表现出更紧密的氢键和 n → π* 相互作用。然而,利用显式水溶解进行的 DFT 和 MD 计算表明,与水的氢键作用受到酰基封端基团立体性的影响。甲酰基封端基团表现出最接近的水-酰胺氢键,而新戊酰基则表现出最长的氢键。在 PDB 中的α-螺旋中,当 N-盖残基为 Gly 时,观察到的紧密酰胺-水氢键频率最高。实验和计算结果相结合表明,N-末端酰胺基团的溶解(水的氢键)是决定α-螺旋稳定性的核心因素。
{"title":"Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability","authors":"Megh R. Bhatt, Himal K. Ganguly and Neal J. Zondlo*, ","doi":"10.1021/acs.biochem.3c00646","DOIUrl":"10.1021/acs.biochem.3c00646","url":null,"abstract":"<p >Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═O<sub><i>i</i></sub>···H<sub><i>i</i>+4</sub>–N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both <i>i</i>/<i>i</i> + 4 hydrogen bonds and <i>i</i>/<i>i</i> + 1 n → π* interactions. This hypothesis was tested in peptides X–AKAAAAKAAAAKAAGY-NH<sub>2</sub>, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and <i>iso</i>-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water–amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide–water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140584933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-15DOI: 10.1021/acs.biochem.4c00056
Josephine C. Ferreon*, Hai Minh Ta, Hyosuk Yun, Kyoung-Jae Choi, My Diem Quan, Phoebe S. Tsoi, Choel Kim, Chul Won Lee* and Allan Chris M. Ferreon*,
NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5–10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.
NANOG 蛋白水平与干细胞多能性相关。NANOG浓度不断波动,NANOG水平低会导致细胞自发分化。以前的文献表明,磷酸化依赖性脯氨酰异构酶 Pin1 是 NANOG 稳定的关键角色。在这里,我们利用核磁共振光谱研究了 Pin1 与 NANOG 非结构化 N 端结构域的分子相互作用,该结构域包含一个带有两个磷酸化位点的 PEST 序列。NANOG PEST 肽的磷酸化增加了与 Pin1 的亲和力。通过系统地增加顺式 PEST 构象的数量,我们发现肽与 Pin1 的脯氨酰异构酶结构域(PPIase)结合得更紧密。两个 PEST 位点的磷酸化和顺式 Pro 增强分别导致 NANOG 与 Pin1 WW 结构域和 PPIase 结构域的结合增加了 5-10 倍。顺式填充的NANOG PEST肽可作为潜在的抑制剂,破坏癌症干细胞中依赖于Pin1的NANOG稳定。
{"title":"Stereospecific NANOG PEST Stabilization by Pin1","authors":"Josephine C. Ferreon*, Hai Minh Ta, Hyosuk Yun, Kyoung-Jae Choi, My Diem Quan, Phoebe S. Tsoi, Choel Kim, Chul Won Lee* and Allan Chris M. Ferreon*, ","doi":"10.1021/acs.biochem.4c00056","DOIUrl":"10.1021/acs.biochem.4c00056","url":null,"abstract":"<p >NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of <i>cis</i> PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and <i>cis</i> Pro enhancement at both PEST sites lead to a 5–10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The <i>cis-</i>populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140584926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}