Biochemistry Biochemistry最新文献

{"title":"Insights into the Activation and Self-Association of Arrestin-1.","authors":"David Salom, Philip D Kiser, Krzysztof Palczewski","doi":"10.1021/acs.biochem.4c00632","DOIUrl":"10.1021/acs.biochem.4c00632","url":null,"abstract":"<p><p>Arrestins halt signal transduction by binding to the phosphorylated C-termini of activated G protein-coupled receptors. Arrestin-1, the first subtype discovered, binds to rhodopsin in rod cells. Mutations in <i>SAG</i>, the gene encoding Arrestin-1, are linked to Oguchi disease, characterized by delayed dark adaptation. Since the discovery of Arrestin-1, substantial progress has been made in understanding the role of these regulatory proteins in phototransduction, including the characterization of visual phenotypes of animals and humans lacking this protein, discovery of splice variants, and documentation of its binding to inositol-polyphosphates. Arrestin-1 was one of the first structurally characterized proteins in the phototransduction cascade. However, there are knowledge gaps regarding the conformational intermediates leading to its binding to phosphorylated rhodopsin. Among various mammalian Arrestin-1 conformations captured via crystallography, the preactivated state is represented by the mutant R175E-Arrestin-1 and by a C-terminally truncated splice variant (p44). This report describes a novel purification method of Arrestin-1 from bovine retinas followed by limited proteolysis to obtain a protein resembling p44. We solved the crystal structure of this preactivated, shortened ^3-367Arrestin-1 at a resolution of 1.40 Å. The structure reveals a more complete picture of the finger loop structure and of the role of the polar core in the activation of Arrestin-1. The structure of ^3-367Arrestin-1 captures an intermediate form halfway between the inactive and fully activated conformations of Arrestin-1. Finally, we addressed the question of Arrestin-1 oligomerization by comparing the packing interfaces in different Arrestin-1 crystals and dimer models predicted by AlphaFold 3.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"364-376"},"PeriodicalIF":2.9,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862518","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":"Metals in Motion: Understanding Labile Metal Pools in Bacteria.","authors":"John D Helmann","doi":"10.1021/acs.biochem.4c00726","DOIUrl":"10.1021/acs.biochem.4c00726","url":null,"abstract":"<p><p>Metal ions are essential for all life. In microbial cells, potassium (K⁺) is the most abundant cation and plays a key role in maintaining osmotic balance. Magnesium (Mg²⁺) is the dominant divalent cation and is required for nucleic acid structure and as an enzyme cofactor. Microbes typically require the transition metals manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn), although the precise set of metal ions needed to sustain life is variable. Intracellular metal pools can be conceptualized as a chemically complex mixture of rapidly exchanging (labile) ions, complemented by those reservoirs that exchange slowly relative to cell metabolism (sequestered). Labile metal pools are buffered by transient interactions with anionic metabolites and macromolecules, with the ribosome playing a major role. Sequestered metal pools include many metalloproteins, cofactors, and storage depots, with some pools redeployed upon metal depletion. Here, I review the size, composition, and dynamics of intracellular metal pools and highlight the major gaps in understanding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"329-345"},"PeriodicalIF":2.9,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11755726/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142929886","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}

{"title":"Inhibition of EphB4 Receptor Signaling by Ephrin-B2-Competitive and Non-Competitive DARPins Prevents Angiogenesis.","authors":"Weronika Trun, Amaury Fernández-Montalván, Yong-Jiang Cao, Bernard Haendler, Dieter Zopf","doi":"10.1021/acs.biochem.4c00431","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00431","url":null,"abstract":"<p><p>The receptor tyrosine kinase EphB4 is involved in tumor angiogenesis, proliferation, and metastasis. Designed ankyrin repeat proteins (DARPins) binding to the EphB4 extracellular domain were identified from a combinatorial library using phage display. Surface plasmon resonance (SPR) allowed us to distinguish between DARPins that either compete with the EphB4 ligand ephrin-B2 for binding to a common site or target a different epitope. The identified DARPins all prevent ligand-induced EphB4 phosphorylation and impair tube formation by endothelial cells in vitro. The competitive DARPin AB1 was additionally shown to inhibit vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-induced angiogenesis in vivo. In summary, we have isolated DARPins that exert antiangiogenic effects by specifically binding to EphB4 and may potentially lead to new cancer therapeutics.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996270","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":"Discovery of Peptidic Siderophore Degradation by Screening Natural Product Profiles in Marine-Derived Bacterial Mono- and Cocultures.","authors":"Mónica Monge-Loría, Weimao Zhong, Nadine H Abrahamse, Stephen Hartter, Neha Garg","doi":"10.1021/acs.biochem.4c00706","DOIUrl":"10.1021/acs.biochem.4c00706","url":null,"abstract":"<p><p>Coral reefs are hotspots of marine biodiversity, which results in the synthesis of a wide variety of compounds with unique molecular scaffolds, and bioactivities, rendering reefs an ecosystem of interest. The chemodiversity stems from the intricate relationships between inhabitants of the reef, as the chemistry produced partakes in intra- and interspecies communication, settlement, nutrient acquisition, and defense. However, the coral reefs are declining at an unprecedented rate due to climate change, pollution, and increased incidence of pathogenic diseases. Among pathogens, <i>Vibrio</i> spp. bacteria are key players resulting in high mortality. Thus, alternative strategies such as application of beneficial bacteria isolated from disease-resilient species are being explored to lower the burden of pathogenic species. Here, we apply coculturing of a coral-derived pathogenic species of <i>Vibrio</i> and beneficial bacteria and leverage recent advancements in untargeted metabolomics to discover engineerable beneficial traits. By chasing chemical change in coculture, we report <i>Microbulbifer</i> spp.-mediated degradation of amphibactins, produced by <i>Vibrio</i> spp. bacteria to sequester iron. Additional biochemical experiments revealed that the degradation occurs in the peptide backbone and requires the enzyme fraction of <i>Microbulbifer</i>. A reduction in iron affinity is expected due to the loss of one Fe(III) binding moiety. Therefore, we hypothesize that this degradation shapes community behaviors as it pertains to iron acquisition, a limiting nutrient in the marine environment, and survival. Furthermore, <i>Vibrio</i> sp. bacteria suppressed natural product synthesis by beneficial bacteria. Understanding biochemical mechanisms behind these interactions will enable engineering probiotic bacteria capable of lowering pathogenic burdens during heat waves and incidence of disease.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142976827","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":"Peptidisc-Assisted Hydrophobic Clustering Toward the Production of Multimeric and Multispecific Nanobody Proteins.","authors":"Yilun Chen, Franck Duong van Hoa","doi":"10.1021/acs.biochem.4c00793","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00793","url":null,"abstract":"<p><p>Multimerization is a powerful engineering strategy for enhancing protein structural stability, diversity and functional performance. Typical methods for clustering proteins include tandem linking, fusion to self-assembly domains and cross-linking. Here we present a novel approach that leverages the Peptidisc membrane mimetic to stabilize hydrophobic-driven protein clusters. We apply the method to nanobodies (Nbs), effective substitutes to traditional antibodies due to their production efficiency, cost-effectiveness and lower immunogenicity, and we demonstrate the formation of multimeric assemblies termed \"polybodies\" (Pbs). Starting with Nbs directed against the green fluorescent protein (GFP), we produce Pbs that display an increased affinity for GFP due to the avidity effect. The benefit of this increased avidity in affinity-based assays is demonstrated with Pbs directed against the human serum albumin. Using the same autoassembly principle, we produce bispecific and auto-fluorescent Pbs, validating our method as a versatile engineering strategy to generate multispecific and multifunctional protein entities. Peptidisc-assisted hydrophobic clustering thus expand the protein engineering toolbox to broaden the scope of protein multimerization in life sciences.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142982258","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":"Journey of PROTAC: From Bench to Clinical Trial and Beyond.","authors":"Kyli Berkley, Julian Zalejski, Nidhi Sharma, Ashutosh Sharma","doi":"10.1021/acs.biochem.4c00577","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00577","url":null,"abstract":"<p><p>Proteolysis-targeting chimeras (PROTACs) represent a transformative advancement in drug discovery, offering a method to degrade specific intracellular proteins. Unlike traditional inhibitors, PROTACs are bifunctional molecules that target proteins for elimination, enabling the potential treatment of previously \"undruggable\" proteins. This concept, pioneered by Crews and his team, introduced the use of small molecules to link a target protein to an E3 ubiquitin ligase, inducing ubiquitination and subsequent degradation of the target protein. By promoting protein degradation rather than merely inhibiting function, PROTACs present a novel therapeutic strategy with enhanced specificity and effectiveness, especially in areas such as cancer and neurodegenerative diseases. Since their initial discovery, the field of PROTAC research has rapidly expanded with numerous PROTACs now designed to target a wide range of disease-relevant proteins. The substantial research, investment, and collaboration across academia and the pharmaceutical industry reflect the growing interest in PROTACs. This Review discusses the journey of PROTACs from initial discovery to clinical trials, highlighting advancements and challenges. Additionally, recent developments in fluorescent and photogenic PROTACs, used for real-time tracking of protein degradation, are presented, showcasing the evolving potential of PROTACs in targeted therapy.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941438","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":"Influence of Magnesium Ions and Crowding Agents on Structure and Stability of RNA Aptamers.","authors":"Jaskirat Kaur, Rajeev Jain, Sumangal Roychowdhury, Rajanya Roy, Krishnananda Chattopadhyay, Ipsita Roy","doi":"10.1021/acs.biochem.4c00468","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00468","url":null,"abstract":"<p><p>Aptamers bind to their targets with exceptional affinity and specificity. However, their intracellular application is hampered by the lack of knowledge about the effect of the cellular milieu on the RNA structure/stability. In this study, cellular crowding was mimicked using polyethylene glycol (PEG), and the crucial role of Mg²⁺ ions in stabilizing the structure of an RNA aptamer was investigated. Increasing the concentration of Mg²⁺ or PEG increased the thermal stability of the aptamer. The crowding effect lowered the requirement of the Mg²⁺ ion to form the binding-competent conformer of the aptamer. This suggests that crowding and other factors may compensate for a lower concentration of Mg²⁺ for proper folding of aptamers inside cells. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing permitted residue-level analysis of the aptamer. Mg²⁺ and/or PEG were shown to be involved in increasing the rigidity or flexibility of different regions of the aptamer. Fluorescence correlation spectroscopy showed a significantly low hydrodynamic radius (<i>R</i>_H) in the presence of molecular crowders and Mg²⁺ ions. We believe that the decreased water activity due to crowding may be responsible for reduced <i>R</i>_H. Our results show that in a crowded environment, the RNA aptamer was exposed to conformers that were not available to it in simple buffer solutions or solely in the presence of lower concentrations of Mg²⁺.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941436","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":"Carbohydrate Deacetylase Unique to Gut Microbe Bacteroides Reveals Atypical Structure.","authors":"Lilith A Schwartz, Jordan O Norman, Sharika Hasan, Olive E Adamek, Elisa Dzuong, Jasmine C Lowenstein, Olivia G Yost, Banumathi Sankaran, Krystle J McLaughlin","doi":"10.1021/acs.biochem.4c00519","DOIUrl":"10.1021/acs.biochem.4c00519","url":null,"abstract":"<p><p><i>Bacteroides</i> are often the most abundant, commensal species in the gut microbiome of industrialized human populations. One of the most commonly detected species is <i>Bacteroides ovatus</i>. It has been linked to benefits like the suppression of intestinal inflammation but is also correlated with some autoimmune disorders, for example irritable bowel disorder (IBD). Bacterial cell surface carbohydrates, like capsular polysaccharides (CPS), may play a role in modulating these varied host interactions. Recent studies have begun to explore the diversity of CPS loci in <i>Bacteroides</i>; however, there is still much unknown. Here, we present structural and functional characterization of a putative polysaccharide deacetylase from <i>Bacteroides ovatus</i> (<i>Bo</i>PDA) encoded in a CPS biosynthetic locus. We solved four high resolution crystal structures (1.36-1.56 Å) of the enzyme bound to divalent cations Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ and performed carbohydrate binding and deacetylase activity assays. Structural analysis of <i>Bo</i>PDA revealed an atypical domain architecture that is unique to this enzyme, with a carbohydrate esterase 4 (CE4) superfamily catalytic domain inserted into a carbohydrate binding module (CBM). Additionally, <i>Bo</i>PDA lacks the canonical CE4 His-His-Asp metal binding motif and our structures show it utilizes a noncanonical His-Asp dyad to bind metal ions. <i>Bo</i>PDA is the first protein involved in CPS biosynthesis from <i>B. ovatus</i> to be characterized, furthering our understanding of significant biosynthetic processes in this medically relevant gut microbe.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"180-191"},"PeriodicalIF":2.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142811426","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}

{"title":"An Alkyne-Containing Isoprenoid Analogue Based on a Farnesyl Diphosphate Scaffold Is a Biologically Functional Universal Probe for Proteomic Analysis.","authors":"Shelby A Auger, Jodi S Pedersen, Sanjay Maity, Andrea M Sprague-Getsy, Ellen L Lorimer, Olivia J Koehn, Steven A Krauklis, Brenna Berns, Katherine M Murphy, Jamal Hussain, Pa Thao, Kaitlyn Bernhagen, Katarzyna Justyna, Anjana P Sundaresan, Daniel B McKim, Carol L Williams, James L Hougland, Ling Li, Mark D Distefano","doi":"10.1021/acs.biochem.4c00558","DOIUrl":"10.1021/acs.biochem.4c00558","url":null,"abstract":"<p><p>Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed <i>in vitro</i> combined with metabolic labeling <i>in cellulo</i>, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated <i>in cellulo</i>, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"138-155"},"PeriodicalIF":2.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11706708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798862","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}

{"title":"A Conserved Lysine in an Ion-Pair with a Catalytic Glutamate Is Critical for U-to-C RNA Editing but Restricts C-to-U RNA Editing.","authors":"Skellie O Chun, Elvin T Garcia, Marcela Rejas, Michael L Hayes","doi":"10.1021/acs.biochem.4c00625","DOIUrl":"10.1021/acs.biochem.4c00625","url":null,"abstract":"<p><p>Plants make pyrimidine base substitutions in organellar mRNAs through the action of sequence-specific nuclear-encoded enzymes. Pentatricopeptide repeat (PPR) proteins are essential for ensuring specificity, while the enzymatic DYW domain is often present at the C-terminus of a PPR protein and dependent on the variant possessing C-to-U and/or U-to-C RNA editing activities. Expression of exogenous DYW-KP variant enzymes in bacteria leads to the modification of RNAs suggestive of U-to-C base changes. The modified RNAs could only be purified from the interphase of an acidic guanidinium thiocyanate-phenol-chloroform experiment. It was projected that in bacteria stable RNA-enzyme cross-links form from a lysyl attack. In this study, RNA editing was examined for dual U-to-C/C-to-U editing enzyme KP6 with conserved lysine residues substituted by alanine. A single lysine was found to be essential for U-to-C editing and, based on the crystal structures of DYW domains, would likely be present in the active site. Crystal structures also suggest that the lysine can potentially form an ion pair with the catalytic glutamate critical for C-to-U RNA editing. Mutation of lysine to alanine greatly stimulated the C-to-U RNA editing by KP6. A ∼319 Da adduct observed on DYW-KP proteins could not be detected on the U-to-C-deficient lysine to alanine point mutant enzymes. This work establishes the critical role for a single lysine in the DYW-KP domain specifically for U-to-C editing activity but also highlights a secondary role for the lysine in modulating C-to-U editing through the formation of an inhibitory ion pair with the catalytic glutamate.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"15-19"},"PeriodicalIF":2.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713852/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798857","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}