Kailash Prasad Prajapati, Masihuzzaman Ansari, Shikha Mittal, Nishant Mishra, Anubhuti Bhatia, Om Prakash Mahato, Bibin Gnanadhason Anand, Karunakar Kar
{"title":"Rapid Coaggregation of Proteins Without Sequence Similarity: Possible Role of Conformational Complementarity.","authors":"Kailash Prasad Prajapati, Masihuzzaman Ansari, Shikha Mittal, Nishant Mishra, Anubhuti Bhatia, Om Prakash Mahato, Bibin Gnanadhason Anand, Karunakar Kar","doi":"10.1021/acs.biochem.4c00282","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00282","url":null,"abstract":"<p><p>Despite extensive research on the sequence-determined self-assembly of both pathogenic and nonpathogenic proteins, the question of how the sequence identity would influence the coassembly or cross-seeding of diverse proteins without distinct sequence similarity remains largely unanswered. Here, we demonstrate that the rapid coaggregation of proteins with negligible sequence similarity is fundamentally governed by preferred heteromeric interactions between their partially unfolded states via the gain of additional charge complementarity and hydrophobic interactions. The partial loss of intramolecular interactions and concurrent gain of non-native intrinsically disordered regions with sticky groups become crucial for both aggressive heteromeric primary nucleation and secondary nucleation events. The results signify the direct relevance of sequence-independent conformational cross-talk between diverse proteins to the foundational events required for the growth of biological multiprotein amyloid deposits.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142405709","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}
Debanjana Maji, Jermaine L Jenkins, Paul L Boutz, Clara L Kielkopf
{"title":"Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions.","authors":"Debanjana Maji, Jermaine L Jenkins, Paul L Boutz, Clara L Kielkopf","doi":"10.1021/acs.biochem.4c00344","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00344","url":null,"abstract":"<p><p><i>De novo</i> mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398689","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}
Per Niklas Hedde, Songning Zhu, Barbara Barylko, Chi-Li Chiu, Luke T Nelson, Michelle A Digman, Joseph P Albanesi, Nicholas G James, David M Jameson
{"title":"Effect of Pathogenic Mutations on the Formation of High-Order Dynamin 2 Assemblies in Living Cells.","authors":"Per Niklas Hedde, Songning Zhu, Barbara Barylko, Chi-Li Chiu, Luke T Nelson, Michelle A Digman, Joseph P Albanesi, Nicholas G James, David M Jameson","doi":"10.1021/acs.biochem.4c00262","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00262","url":null,"abstract":"<p><p>Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues <sub>555</sub>DEE<sub>557</sub> (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2<sup>ΔDEE</sup> and CNM-linked DNM2<sup>A618T</sup> mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2<sup>WT</sup>). However, DNM2<sup>A618T</sup> forms cytoplasmic inclusions at concentrations lower than those of either DNM2<sup>WT</sup> or DNM2<sup>ΔDEE</sup>, suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398688","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}
Saba Abbasi Dezfouli, Marcel E Michailides, Hasan Uludag
{"title":"Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases.","authors":"Saba Abbasi Dezfouli, Marcel E Michailides, Hasan Uludag","doi":"10.1021/acs.biochem.4c00327","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00327","url":null,"abstract":"<p><p>Hematological disorders result in significant health consequences, and traditional therapies frequently entail adverse reactions without addressing the root cause. A potential solution for hematological disorders characterized by gain-of-function mutations lies in the emergence of small interfering RNA (siRNA) molecules as a therapeutic option. siRNAs are a class of RNA molecules composed of double-stranded RNAs that can degrade specific mRNAs, thereby inhibiting the synthesis of underlying disease proteins. Therapeutic interventions utilizing siRNA can be tailored to selectively target genes implicated in diverse hematological disorders, including sickle cell anemia, β-thalassemia, and malignancies such as lymphoma, myeloma, and leukemia. The development of efficient siRNA silencers necessitates meticulous contemplation of variables such as the RNA backbone, stability, and specificity. Transportation of siRNA to specific cells poses a significant hurdle, prompting investigations of diverse delivery approaches, including chemically modified forms of siRNA and nanoparticle formulations with various biocompatible carriers. This review delves into the crucial role of siRNA technology in targeting and treating hematological malignancies and disorders. It sheds light on the latest research, development, and clinical trials, detailing how various pharmaceutical approaches leverage siRNA against blood disorders, mainly concentrating on cancers. It outlines the preferred molecular targets and physiological barriers to delivery while emphasizing the growing potential of various therapeutic delivery methods. The need for further research is articulated in the context of overcoming the shortcomings of siRNA in order to enrich discussions around siRNA's role in managing blood disorders and aiding the scientific community in advancing more targeted and effective treatments.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398687","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}
Shweta Shree, Mark A McLean, Andrew G Stephen, Stephen G Sligar
{"title":"KRas4b-Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics.","authors":"Shweta Shree, Mark A McLean, Andrew G Stephen, Stephen G Sligar","doi":"10.1021/acs.biochem.4c00116","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00116","url":null,"abstract":"<p><p>KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386374","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":"Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.","authors":"Shabnam, Rajiv Bhat","doi":"10.1021/acs.biochem.4c00362","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00362","url":null,"abstract":"<p><p>The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most <i>in vitro</i> studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386373","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}
Xingguo R Chen, Ana Y Mercedes-Camacho, Kimberly A Wilson, Jill J Bouchard, Jeffrey W Peng, Felicia A Etzkorn
{"title":"Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent.","authors":"Xingguo R Chen, Ana Y Mercedes-Camacho, Kimberly A Wilson, Jill J Bouchard, Jeffrey W Peng, Felicia A Etzkorn","doi":"10.1021/acs.biochem.4c00231","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00231","url":null,"abstract":"<p><p>Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-<i>cis/trans</i>-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R<sup>1</sup>CO-pSer-Pro-NHR<sup>2</sup>, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with <sup>15</sup>N-labeled Pin1 were used to measure <i>K</i><sub>d</sub> for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386375","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}
Tomotsumi Fujisawa, Nozomi Tanaka, Jun Tamogami, Masashi Unno
{"title":"Retinal Chromophore Configuration in the O Intermediate of Sensory Rhodopsin II from <i>Natronomonas pharaonis</i>.","authors":"Tomotsumi Fujisawa, Nozomi Tanaka, Jun Tamogami, Masashi Unno","doi":"10.1021/acs.biochem.4c00420","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00420","url":null,"abstract":"<p><p>Sensory rhodopsin II (SRII) is a prototype photosensor that binds the retinal Schiff base chromophore. Upon photoabsorption, SRII is transformed into the signaling state, where two long-lived photointermediates are known to contribute. One is the M intermediate containing the deprotonated 13-<i>cis</i> chromophore, and the other is the O intermediate that is believed to have the protonated all-<i>trans</i> chromophore. The chromophore in the O intermediate is also thought to have the atypical 15-<i>syn</i> (C═N <i>cis</i>) configuration about the Schiff base moiety. In this communication, we study SRII from <i>Natronomonas pharaonis</i> (<i>Np</i>SRII) using Raman spectroscopy and find that the retinal chromophore configuration in the O intermediate is the 13-<i>cis</i>, 15-<i>anti</i> (C═N <i>trans</i>), contrary to the conventional notion. This result points out the revision of the chromophore structural changes underlying the long-lived signaling state of SRII.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386376","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":"Atomistic Insights into Sequence-Mediated Spontaneous Association of Short RNA Chains.","authors":"Manas Mondal, Yi Qin Gao","doi":"10.1021/acs.biochem.4c00293","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00293","url":null,"abstract":"<p><p>RNA-RNA association and phase separation appear to be essential for the assembly of stress granules and underlie RNA foci formation in repeat expansion disorders. RNA molecules are found to play a significant role in gene-regulatory functions via condensate formation among themselves or with RNA-binding proteins. The interplay between driven versus spontaneous processes is likely to be an important factor for controlling the formation of RNA-mediated biomolecular condensate. However, the sequence-specific interactions and molecular mechanisms that drive the spontaneous RNA-RNA association and help to form RNA-mediated phase-separated condensate remain unclear. With microseconds-long atomistic molecular simulations here, we report how essential aspects of RNA chains, namely, base composition, metal ion binding, and hydration properties, contribute to the association of the series of simplest biologically relevant homopolymeric and heteropolymeric short RNA chains. We show that spontaneous processes make the key contributions governed by the sequence-intrinsic properties of RNA chains, where the definite roles of base-specific hydrogen bonding and stacking interactions are prominent in the association of the RNA chains. Purine versus pyrimidine contents of RNA chains can directly influence the association properties of RNA chains by modulating hydrogen bonding and base stacking interactions. This study determines the impact of ionic environment in sequence-specific spontaneous association of short RNA chains, hydration features, and base-specific interactions of Na<sup>+</sup>, K<sup>+</sup>, and Mg<sup>2+</sup> ions with RNA chains.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386372","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}
Ana C Tan, Patrick S Irving, Jordan T Koehn, Shouhong Jin, David Y Qiu, Kevin M Weeks
{"title":"Fingerprinting Tertiary Structure in Complex RNAs Using Single-Molecule Correlated Chemical Probing.","authors":"Ana C Tan, Patrick S Irving, Jordan T Koehn, Shouhong Jin, David Y Qiu, Kevin M Weeks","doi":"10.1021/acs.biochem.4c00343","DOIUrl":"10.1021/acs.biochem.4c00343","url":null,"abstract":"<p><p>Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142363346","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}