Archives of biochemistry and biophysics最新文献

{"title":"Decoding KRAS Dynamics: Exploring the Impact of Mutations and Inhibitor Binding.","authors":"Divya Pandey, Kuldeep K Roy","doi":"10.1016/j.abb.2024.110279","DOIUrl":"https://doi.org/10.1016/j.abb.2024.110279","url":null,"abstract":"<p><p>KRAS (Kirsten rat sarcoma viral oncogene homologue), the most common mutated protein in human cancers, is the leading cause of morbidity and mortality. Before Sotorasib (AMG-510) was approved for non-small cell lung cancer treatment in 2020, the oncogenic KRAS mutations were believed to be non-druggable. High-resolution X-ray crystal structures of GDP-bound KRAS mutants with and without inhibitor resolved. Nevertheless, to develop inhibitors targeting oncogenic KRAS mutants, understanding the dynamics of protein conformations and respective binding sites is crucial. In the present study, multiple molecular dynamics (MD) simulations were conducted on wild-type and mutant KRAS structures to understand how G12C or G12D mutations lead to the stabilization of the active state and how KRAS inhibitors lock the mutated conformations in their inactive state. The study found that the guanosine diphosphate (GDP)-bound KRAS mutants, G12C and G12D, were locked in the inactive state, in terms of stability, when the KRAS inhibitors, AMG-510 and MRTX1133, respectively, bind to the respective Switch-II (S-II) pocket. Covalent inhibitor AMG-510 locked the inactive GDP-bound KRAS^G12C mutant more efficiently when compared to the non-covalent inhibitor MRTX1133. The Cα atom distance between key highly dynamic amino acids from P-loop, Switch-I, and Switch-II domains, lying within 4Å of the ligand, were stable in the KRAS mutant with bound inhibitors (AMG-510 or MRTX1133), but were dynamic in the absence of any inhibitor throughout the microsecond simulation. According to the per-residue energy decomposition results, S-II amino acids in inhibitor-free KRAS^G12C and KRAS^G12D mutants showed higher variations in energy values as compared to AMG-510-bound KRAS^G12C and MRTX1133-bound KRAS^G12D, respectively. For example, the inhibitor-free KRAS^G12C exhibited higher variations in energy values in the S-II residues, namely, Thr58, Gln61, Glu63, and Arg68, as compared to the AMG-510-bound KRAS^G12C. The study found that the higher stability of AMG-510 in torsion angles was due to its covalent nature of binding to the KRAS^G12C mutant. The S-II amino acids, namely, Thr58, Glu63, and Arg68 remained stable in AMG-510-bound KRAS^G12C. The study showed that AMG-510 binding significantly stabilizes the amino acids surrounding it, surpassing that of MRTX1133. The insights gained in the present study is expected to be useful in the design and development of new KRAS-targeted drugs.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110279"},"PeriodicalIF":3.8,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142875601","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}

{"title":"Kinetic studies of bifurcating flavoproteins.","authors":"Russ Hille, Dimitri Niks, Wayne Vigil, Jessica Tran, Steve Ortiz, Kevin Menjivar, Derek Nguyen","doi":"10.1016/j.abb.2024.110278","DOIUrl":"10.1016/j.abb.2024.110278","url":null,"abstract":"<p><p>Since their original proposal in 2008, a number of broadly distributed flavoprotein systems catalyzing electron bifurcation have been identified that play key roles in the bioenergetics of anaerobic bacteria and archaea. While the overall thermodynamics of flavin-based electron bifurcation are now quite well-understood, the same cannot be said of their kinetic behavior. The present account represents a summary of results obtained with several electron-electron bifurcating systems, shamelessly focusing on work done in the authors' laboratory.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110278"},"PeriodicalIF":3.8,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871095","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}

{"title":"Exosomal signaling in cancer metastasis: Molecular insights and therapeutic opportunities.","authors":"Manasi S Pote, Rajesh N Gacche","doi":"10.1016/j.abb.2024.110277","DOIUrl":"10.1016/j.abb.2024.110277","url":null,"abstract":"<p><p>Exosomes are membrane-bound extracellular vesicles that play a role in exchanging biological products across membranes and serve as intermediaries in intercellular communication to maintain normal homeostasis. Numerous molecules, including lipids, proteins, and nucleic acids are enclosed in exosomes. Exosomes are constantly released into the extracellular environment and exhibit distinct characteristics based on the secreted cells that produce them. Exosome-mediated cell-to-cell communication has reportedly been shown to affect multiple cancer hallmarks, such as immune response modulation, pre-metastatic niche formation, angiogenesis, stromal cell reprogramming, extracellular matrix architecture remodeling, or even drug resistance, and eventually the development and metastasis of cancer cells. Exosomes can be used as therapeutic targets and possible diagnostic biomarkers by selectively loading oncogenic molecules into them. We highlight the important roles that exosomes play in cancer development in this review, which may lead to the development of fresh approaches for future clinical uses.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110277"},"PeriodicalIF":3.8,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871093","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}

{"title":"Altering substrate specificity of a thermostable bacterial monoamine oxidase by structure-based mutagenesis.","authors":"Lorenzo Basile, Chiara Poli, Lars L Santema, Răzvan C Lesenciuc, Marco W Fraaije, Claudia Binda","doi":"10.1016/j.abb.2024.110276","DOIUrl":"https://doi.org/10.1016/j.abb.2024.110276","url":null,"abstract":"<p><p>Bacterial monoamine oxidases (MAOs) are FAD-dependent proteins catalyzing a relevant reaction for many industrial biocatalytic applications, ranging from production of enantiomerically pure building blocks for pharmaceutical synthesis to biosensors for monitoring food and beverage quality. The thermostable MAO enzyme from Thermoanaerobacterales bacterium (MAO_Tb) is about 36% identical to both putrescine oxidase and human MAOs and can be efficiently produced in Escherichia coli. MAO_Tb preferentially acts on n-alkyl monoamines but shows detectable activity also on polyamines and aromatic monoamines. The crystal structures of MAO_Tb in complex with putrescine, benzylamine, spermidine and n-heptylamine at resolution ranging from 1.6 to 2.3 Å resolution revealed the binding mode of substrates to the enzyme. The MAO_Tb active site is highly conserved in the inner part of the cavity in front of the flavin ring (re face), where the presence of two tyrosine residues creates the substrate amine binding site that is found also in human MAOs. Instead, more distantly from the flavin, the entrance of the catalytic site is much more open in MAO_Tb and features a different arrangement of amino acids. Site-directed mutagenesis targeting residues Ala168, Thr199 and Val324 allowed the identification of key residues in ligand binding to alter substrate specificity. The A168D variant showed a higher activity on putrescine than wild-type, whereas by replacing either Thr199 or Val324 to Trp a marked enhancement in k_cat/K_M values was found on n-alkyl-monoamines and on aromatic amines.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110276"},"PeriodicalIF":3.8,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871091","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}

{"title":"Structural and functional snapshots of a broad-specificity endoglucanase from Thermogutta terrifontis for biomass saccharification.","authors":"Naveed Hussain, Halina Mikolajek, Peter J Harrison, Neil Paterson, Muhammad W Akhtar, Saima Sadaf, James H Naismith","doi":"10.1016/j.abb.2024.110274","DOIUrl":"10.1016/j.abb.2024.110274","url":null,"abstract":"<p><p>Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-d-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70 to 80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80°C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)₈-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110274"},"PeriodicalIF":3.8,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862911","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}

{"title":"MiR-495 reverses in the mechanical unloading, random rotating and aging induced muscle atrophy via targeting MyoD and inactivating the Myostatin/TGF-β/Smad3 axis.","authors":"Chenyan Zhang, Yile Tian, Xinli Liu, Xuezhou Yang, Shanfeng Jiang, Ge Zhang, Changqing Yang, Wenjing Liu, Weihong Guo, Wenzhe Zhao, Dachuan Yin","doi":"10.1016/j.abb.2024.110273","DOIUrl":"10.1016/j.abb.2024.110273","url":null,"abstract":"<p><p>Mechanical unloading can lead to homeostasis imbalance and severe muscle disease, in which muscle atrophy was one of the disused diseases. However, there were limited therapeutic targets for such diseases. In this study, miR-495 was found dramatically reduced in atrophic skeletal muscle induced by mechanical unloading models both in vitro and in vivo, including the random positioning model (RPM), tail-suspension (TS) model, and aged mice model. Enforced miR-495 expression by its mimic could enormously facilitate the differentiation and regeneration of both mouse myoblast C2C12 cells and muscle satellite cells. Furthermore, MyoD was proved as the directly interacted gene of miR-495, and their interaction was crucial for myotube formation. Enforced miR-495 expression could intensively strengthen the muscle mass, in situ muscular electrophysiological indexes, including peak tetanic tension (Po) and peak twitch tension (Pt), and the cross-sectional areas (CSA) of muscle fibers via targeting MyoD and inactivating the Myostatin/TGF-β/Smad3 signaling pathway, indicating that miR-495 can be proposed as an effective target for muscle atrophy treatment induced by in the mechanical unloading, random rotating and aging.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110273"},"PeriodicalIF":3.8,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862909","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}

{"title":"IN SILICO BASED RE-ENGINEERING OF A COMPUTATIONALLY DESIGNED BIOSENSOR WITH ALTERED SIGNALLING MODE AND IMPROVED DYNAMIC RANGE.","authors":"Dustin D Smith, D Wade Abbott, Hans-Joachim Wieden","doi":"10.1016/j.abb.2024.110275","DOIUrl":"https://doi.org/10.1016/j.abb.2024.110275","url":null,"abstract":"<p><p>A current challenge in the rational design of biomolecular sensors is the ability to custom design binding affinities and detection mode in silico. To this end, we re-engineered a previously reported computationally-designed fluorescent maltooligosaccharide (MOS)-detecting biosensor to both alter its ligand-binding affinity and to analyse the underlying sensing mechanism. The dynamic range of the biosensor was expanded through the computer aided introduction of a series of amino acid substitutions in the starting protein scaffold (MalX from Streptococcus pneumoniae), which generated a biosensor set with binding affinities spanning over five orders of magnitude. The impact of the introduced substitutions on the underlying mode of signal generation was assessed in silico using our previously reported Computational Identification of Non-disruptive Conjugation sites (CINC) pipeline. CINC utilizes molecular dynamics simulations and an in-house developed algorithm to examine and exploit the structural dynamics of a protein at amino acid-level resolution. Using CINC, we demonstrate that re-engineering of the MOS-detecting biosensor set resulted in sensors with two distinct output modes which differed based on local conformational changes at the fluorescently modified reporter position. These output modes were classified as \"ligand-sensing\"-type biosensors (readout based on the tool sensing a unique conformation in the ligand-bound state), and \"apo-sensing\"-type biosensors (readout based on the tool sensing a unique conformation in the apo state). Together, these results demonstrate that structural dynamics at the individual amino acid residue level can be used as an engineer-able feature to rationally alter the fluorescence reporting properties of a biosensing device. Moving forward, the CINC workflow can also be adapted for the rational design of protein dynamic properties maximizing its utility as an in silico design platform for custom biomolecular tools.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110275"},"PeriodicalIF":3.8,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142851701","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}

{"title":"Molecular characterization of the E2 conjugating enzyme LinfUbc13 in Leishmania infantum.","authors":"Eduardo Vagner Rodrigues da Silva, Caroline Torres, Hariel Nemamiah Escolarique Ribeiro, Camila Rolemberg Santana Travaglini Berti de Correia, Taissa de Oliveira de Castro, Giovanna da Costa Mancin, Mayla Gabriela Zanchetta Venancio, Munira Muhammad Abdel Baqui, Felipe Roberti Teixeira, Marcelo Damário Gomes","doi":"10.1016/j.abb.2024.110272","DOIUrl":"10.1016/j.abb.2024.110272","url":null,"abstract":"<p><p>UBC13 is an orthologue of Homo sapiens ubiquitin-conjugation E2 enzymes described in Leishmania mexicana, a null mutant lacking this gene cannot be produced, suggesting essential functions in this parasite. Leishmania infantum is an etiological agent of visceral leishmaniasis, the most severe type of disease that is potentially fatal if untreated. The ubiquitination process has been targeted for leishmanicidal compounds, indicating its essential function in parasite homeostasis. Therefore, the molecular characterization of the ubiquitination process may provide a better understanding of the molecular and cellular basis of leishmaniasis. Here, we characterized the gene LINF_350017900 in Leishmania infantum, which was named LinfUBC13, an E2 orthologue of UBC13 in Leishmania mexicana and the UBE2D family in Homo sapiens, sharing 72-74 % identity with UBE2D1, UBE2D2, and UBE2D3. LinfUbc13 contains conserved catalytic residues, including Cys86 and the HPN motif, which are essential for ubiquitin-conjugating activity. Structural analysis revealed a high similarity between LinfUbc13 and human UBE2D proteins, with a root-mean-square deviation (RMSD) of 0.4 Å, suggesting conserved functions. Recombinant LinfUbc13 was expressed and shown to accept ubiquitin from E1, forming a thioester intermediate. Functional assays demonstrated that LinfUbc13 transfers ubiquitin to p53 through human HDM2 E3 ligase, confirming its role in ubiquitination. Subcellular localization showed that LinfUbc13 was distributed throughout the parasite cytoplasm. These findings highlight the conserved nature of the ubiquitin-proteasome system between Leishmania infantum and Homo sapiens, showing that LinfUbc13 is an E2 enzyme that plays a crucial role in parasitic development.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110272"},"PeriodicalIF":3.8,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845690","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}

{"title":"Structural and biophysical characterization of the cytoplasmic domains of HprS kinase and its interactions with the cognate regulator HprR.","authors":"Anna Koczurowska, David Ruiz Carrillo, María García Alai, Małgorzata Zakłos-Szyda, Grzegorz Bujacz, Agnieszka J Pietrzyk-Brzezinska","doi":"10.1016/j.abb.2024.110269","DOIUrl":"10.1016/j.abb.2024.110269","url":null,"abstract":"<p><p>The HprSR constitutes the bacterial two-component regulatory system engaged by Escherichia coli to reduce the damaging effects of reactive chlorine and oxygen species present in its cytosol. Hypochlorous acid (HOCl) has been shown to be the molecule capable of activating of the HprSR system. HOCl is produced upon pathogen invasion by phagocytic cells of the human innate immune system, particularly neutrophils, to take advantage of its powerful antimicrobial attributes. Therefore, comprehensive studies concerning bacterial sensing and regulatory HprSR system are indispensable in understanding and effectively eliminating pathogens. Here we present the first crystal structure, solved at 1.7 Å resolution, of the HprS cytoplasmic domains arranged as a homodimer. In both protomers, the catalytic ATP-binding domain contains a non-hydrolysable ATP analog coordinated by a magnesium ion. This structure allowed us to provide a detailed characterization of kinase-substrate interaction. Furthermore, the structural data are supported by biophysical studies of kinase interaction with cognate response regulator HprR and substrate ATP. The kinase activity is also assessed in the presence or absence of HprR.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110269"},"PeriodicalIF":3.8,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142833763","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}

{"title":"High stability of the radical at the catalytic center of cytochrome c oxidase.","authors":"Adriana Tomkova, Erik Cizmar, Daniel Jancura, Marian Fabian","doi":"10.1016/j.abb.2024.110271","DOIUrl":"https://doi.org/10.1016/j.abb.2024.110271","url":null,"abstract":"<p><p>In aerobic organisms, cellular respiration is associated with electron transfer through a respiratory system of membrane-bound complexes. This electron flow is terminated by the reduction of dioxygen to water by respiratory oxidases. Cytochrome c oxidase (CcO) is a widely distributed heme-copper-oxygen reductase (HCO) found in all mitochondria and some bacteria. However, the sequential reduction of O₂ to water in CcO generates a protein-based radical at the catalytic heme a₃-Cu_B site. To avoid the potential damage from the radical, CcO has apparently developed protective mechanisms. Protection by transfer of the highly oxidizing equivalent over considerable distances away from the catalytic site by redox-active Tyr/Trp chains has been previously demonstrated in bovine CcO. However, the rate of the radical migration from the catalytic center has not yet been determined for any HCO. In this work, we show that the radical escapes from the catalytic center of the ferryl P_M intermediate of bovine CcO within minutes, which is much longer than the time of its functional reduction during cellular respiration. Apparently, this high stability has evolved to avoid the dissipation of energy released during the oxygen reduction with substrate electrons.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110271"},"PeriodicalIF":3.8,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845689","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}