Central Domain Research Articles

Network dynamics in subjective well-being and their differences across age groups.

Although the structure of subjective well-being (SWB) has been examined in various studies, no consensus on its structure has yet been reached. This may be due to a neglect of the construct's dynamic aspects and domain satisfaction as a core aspect of SWB. This article aimed to overcome existing research gaps by applying network modeling to longitudinal data of 32,700 adults (24-64 years old) from the German Socioeconomic Panel to analyze within- and between-person dynamics in the structure of SWB across the lifespan. Results indicated that the relationships across SWB components differed across the investigated within- and between-person network structures. Family, work, and income satisfaction tended to be the most central domains across different levels of analysis. The relationship between life and domain satisfaction was neither solely top-down nor bottom-up but instead characterized by distinct, mostly reciprocal relationships. Furthermore, the dynamic relationships of SWB were similar across compared age groups. In sum, the results suggest that the structure of SWB differs between the within-person level and the between-person level but does not change fundamentally throughout middle adulthood. Additionally, this study demonstrates the importance of considering domain satisfaction as an essential component of SWB and that psychometric network models can advance our understanding of the structure and dynamics of SWB. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Structural diversity and distribution of NMCP-class nuclear lamina proteins in streptophytic algae.

Nuclear Matrix Constituent Proteins (NMCPs) in plants function like animal lamins, providing the structural foundation of the nuclear lamina and regulating nuclear organization and morphology. Although they are well-characterized in angiosperms, the presence and structure of NMCPs in more distantly related species, such as streptophytic algae, are relatively unknown. The rapid evolution of NMCPs throughout the plant lineage has caused a divergence in protein sequence that makes similarity-based searches less effective. Structural features are more likely to be conserved compared to primary amino acid sequence; therefore, we developed a filtration protocol to search for diverged NMCPs based on four physical characteristics: intrinsically disordered content, isoelectric point, number of amino acids, and the presence of a central coiled-coil domain. By setting parameters to recognize the properties of bona fide NMCP proteins in angiosperms, we filtered eight complete proteomes from streptophytic algae species and identified strong NMCP candidates in six taxa in the Classes Zygnematophyceae, Charophyceae, and Klebsormidophyceae. Through analysis of these proteins, we observed structural variance in domain size between NMCPs in algae and land plants, as well as a single block of amino acid conservation. Our analysis indicates that NMCPs are absent in the Mesostigmatophyceae. The presence versus absence of NMCP proteins does not correlate with the distribution of different forms of mitosis (e.g., closed/semi-closed/open) but does correspond to the transition from unicellularity to multicellularity in the streptophytic algae, suggesting that an NMCP-based nucleoskeleton plays important roles in supporting cell-to-cell interactions.

Abstract 4137543: Prevalence, Genetic Correlates and Prognostic Significance of an Apical Pseudo-Infarct Late Gadolinium Enhancement Pattern in LMNA -Mediated Cardiac Laminopathy

Background: Disease-causative variants in LMNA -encoded lamin A/C cause a genetic cardiomyopathy characterized by atrioventricular (AV) block, atrial fibrillation (AF) and ventricular arrhythmias (VA). Whether domain/variant-specific late gadolinium enhancement (LGE) patterns exist and carry prognostic significance is unclear. Objective: To determine if specific LGE patterns correlate with LMNA variant type/missense variant localization and clinical outcomes in a large cohort of cardiac laminopathy patients. Methods: Retrospectively, 621 genotype-positive arrhythmogenic/dilated cardiomyopathy patients were analyzed to identify those with a disease-causative variant in LMNA -encoded lamin A/C. After exclusion of patients without cardiac magnetic resonance data, pertinent demographic/clinical data was extracted from the electronic medical record. LGE distribution (subepicardial, midmyocardial, subendocardial, transmural) and location (septal, free wall, apical) patterns were analyzed by variant type (missense vs truncating) and missense variant localization (central rod domain vs tail domain) and correlated with advanced heart failure (AHF), MVA, AV block, AF and thromboembolic (TE) events. Results: 70 out of 116 (60%) LMNA variant-positive (mean age 39±12, 43% male and 94% Caucasian) were included. Missense variants localizing to the C-terminal tail region showed a higher prevalence of transmural and apical LGE (Table). Amongst the 5 (7%) patients with this distinct apical pseudo-infarct LGE pattern, p.Arg471His-LMNA was identified in all cases. Among the 48/70 (69%) of patients with clinically manifest disease, p.Arg471His-LMNA-positive patients (n=6) were noted to have an increased prevalence of TE events [3 (50%) vs 5 (12%), 0=0.050] despite a reduced prevalence of AF [2 (33%) vs 32 (76%), p= 0.031]. No difference in AHF or MVA were observed, but AV block was less prevalent [1 (20%) vs 29 (74%), p= 0.029]. Conclusion: p.Arg471His-LMNA causes a distinct apical pseudo-infarct LGE pattern associated with an increased risk of TE events and a decreased prevalence of AV block and AF. Future studies are needed to better refine the role of domain/variant-specific risk-stratification/anticoagulation in cardiac laminopathy.

GPCR-like Protein ZmCOLD1 Regulate Plant Height in an ABA Manner.

G protein-coupled receptors (GPCRs) are sensors for the G protein complex to sense changes in environmental factors and molecular switches for G protein complex signal transduction. In this study, the homologous gene of GPCR-like proteins was identified from maize and named as ZmCOLD1. Subcellular analysis showed that the ZmCOLD1 protein is localized to the cell membrane and endoplasmic reticulum. A CRISPR/Cas9 knock-out line of ZmCOLD1 was further created and its plant height was significantly lower than the wild-type maize at both the seedling and adult stages. Histological analysis showed that the increased cell number but significantly smaller cell size may result in dwarfing of zmcold1, indicating that the ZmCOLD1 gene could regulate plant height development by affecting the cell division process. Additionally, ZmCOLD1 was verified to interact with the maize Gα subunit, ZmCT2, though the central hydrophilic loop domain by in vivo and in vitro methods. Abscisic acid (ABA) sensitivity analysis by seed germination assays exhibited that zmcold1 were hypersensitive to ABA, indicating its important roles in ABA signaling. Finally, transcriptome analysis was performed to investigate the transcriptional change in zmcold1 mutant. Overall, ZmCOLD1 functions as a GPCR-like protein and an important regulator to plant height.

Geological and mining mapping approach by coupling geological and geophysical field data: application to the Central Domain of the Mbere division (Adamawa-Cameroon)

This study is based on the coupling of geological field data with geophysical studies in the geological mapping of the Central Domain of the Mbere Division (Adamawa-Cameroon). The first step was to calculate the residual grid associated with the geophysical data that will be used for the mapping of the basement. The second step before mapping consisted in calculating the analytical signal associated with the residual grid, used to interpret the geological formations under cover or in intrusion. Subsequently, a geological mapping approach based on the categorisation of geophysical signatures into ranges using the Encom Discover program was adopted. This approach is based on the analysis of the relief of geophysical anomalies. The process of categorisation which is done iteratively, takes into consideration in the same georeferenced space, the resolution of the input data, the variation of the field considered and the structural model previously interpreted. The result of the categorisation is then compared to the spatial distribution of outcrop data to build the geological model. The application of this approach to the Central Domain of the Mbere Division led to the production of a synthetic geological map at a scale of 1/75,000. The superposition of this map to the topographic model allows to observe a concordance between the Cretaceous basins represented by their edge faults and the topographic depression zones. The various mineral indicators (primary and secondary) collected during the fieldwork, combined with the geological model (1:75,000 scale) as well as the Cretaceous deposits and vein intrusion models were used to create the mineral indicators and targets map for this sector. All these models can thus be used to underpin mineral exploration projects in the study area.

Molecularly Imprinted Polymers for Highly Specific Bioorthogonal Catalysis Inside Cells.

Transition metal catalysts (TMCs) mediated bioorthogonal catalysis expand the chemical possibilities within cells. Developing synthetic TMCs tools that emulate the efficiency and specificity of natural metalloenzymes is a rewarding yet challenging endeavor. Here, we highlight the potential of molecularly imprinted enzyme mimics (MIEs) containing a Cu center and specific substrate binding domain, for conducing dimethylpropargyloxycarbonyl (DmProc) cleavage reactions within cells. Our studies reveal that the Cu-MIEs act as highly specific guides, precisely catalyzing target substrates, even in glutathione (GSH)-rich cellular environments. By adapting templates similar to the target substrates, we evolved Cu-MIEs activity to a high level and provided a method to broaden its scope to other unique substrates. This system was applied to a thyroid hormone (T3)-responsive gene switch model, inducing firefly luciferase expression by T3 in cells. This approach verifies that MIEs effectively rescue DmProc-bearing T3 prodrugs and seamlessly integrating themself into cellular biocatalytic networks.

Molecularly Imprinted Polymers for Highly Specific Bioorthogonal Catalysis Inside Cells

AbstractTransition metal catalysts (TMCs) mediated bioorthogonal catalysis expand the chemical possibilities within cells. Developing synthetic TMCs tools that emulate the efficiency and specificity of natural metalloenzymes is a rewarding yet challenging endeavor. Here, we highlight the potential of molecularly imprinted enzyme mimics (MIEs) containing a Cu center and specific substrate binding domain, for conducing dimethylpropargyloxycarbonyl (DmProc) cleavage reactions within cells. Our studies reveal that the Cu‐MIEs act as highly specific guides, precisely catalyzing target substrates, even in glutathione (GSH)‐rich cellular environments. By adapting templates similar to the target substrates, we evolved Cu‐MIEs activity to a high level and provided a method to broaden its scope to other unique substrates. This system was applied to a thyroid hormone (T3)‐responsive gene switch model, inducing firefly luciferase expression by T3 in cells. This approach verifies that MIEs effectively rescue DmProc‐bearing T3 prodrugs and seamlessly integrating themself into cellular biocatalytic networks.

Interaction of PGC7 and HP1BP3 Maintains Meg3-DMR Methylation by Regulating Chromatin Configuration.

Genomic imprinting is essential for mammalian development. PGC7, an important maternal factor, binds to dimethylated histone H3K9 (H3K9me2), maintaining DNA methylation in zygotes and stem cells. However, the underlying molecular mechanisms of PGC7-maintained genomic imprinting in stem cells are not clear. Our previous study has identified that PGC7 interacts with HP1BP3, a novel member of the histone H1 family. In this study, we found that PGC7 interacts with the central globular domain of HP1BP3 through its C-terminal tail and that HP1BP3 is responsible for the recruitment of PGC7 at the Meg3 differentially methylated region (DMR) in the Dlk1-Dio3 imprinted domain. HP1BP3 or PGC7 depletion decreases enrichment in the Meg3-DMR, leading to DNA hypermethylation in this region. Moreover, the cooperative binding of PGC7 and HP1BP3 can antagonize the enrichment of DNMT3A in the Meg3-DMR, and the depletion of HP1BP3 or PGC7 separately induces chromosome decondensation in this region. In summary, this is the first study demonstrating that PGC7 and HP1BP3 synergistically maintain the methylation status of the Meg3-DMR by enabling a chromatin configuration that interferes with the binding of the de novo DNA methyltransferase DNMT3A.

Cryo-electron microscopy structure of the di-domain core of Mycobacterium tuberculosis polyketide synthase 13, essential for mycobacterial mycolic acid synthesis.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis, the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

Involvement of BK Channels and Ryanodine Receptors in Salicylate-induced Tinnitus.

Neural hyperexcitability of the central auditory system is a key pathological characteristic of tinnitus, but its underlying molecular mechanisms remain elusive. The large-conductance Ca2+-activated K+ channel (BK) plays a crucial role in down- or upregulating neuronal activity. This study aims to investigate the role of BK channels in mediating tinnitus-associated neural hyperexcitability and elucidate the mechanisms behind it. Immunofluorescent staining revealed extensive expression of the BK channels on neurons within the central auditory system of rats. After long-term systemic administration of salicylate, a stable tinnitus inducer, we observed a significant change in the expression levels of BKα and β4 subunits in the rat central auditory system. In addition, salicylate was found to enhance the outward potassium currents mediated by the BK channel when exogenously expressed in HEK293 cells. Interestingly, this effect could be blocked by ryanodine, a potent inhibitor of ryanodine receptors (RyRs). Molecular docking identified Gln4020 within the central domain of RyR as a key residue in RyR-salicylate interactions. The results indicated that salicylate might directly activate RyRs leading to Ca2+ release from endoplasmic reticulum, and increased BK currents subsequently. Systemic treatment with paxilline, a potent blocker of BK channel, selectively reversed the increased P4/P1 amplitude ratios in the frequency region of tinnitus perception induced by single-dose salicylate administration. These results suggest that BK channels and ryanodine receptors may play a selective role in salicylate-induced tinnitus.

Coupling of Spectrin Repeat Modules for the Assembly of Nanorods and Presentation of Protein Domains.

Modular protein engineering is a powerful approach for fabricating high-molecular-weight assemblies and biomaterials with nanoscale precision. Herein, we address the challenge of designing an extended nanoscale filamentous architecture inspired by the central rod domain of human dystrophin, which protects sarcolemma during muscle contraction and consists of spectrin repeats composed of three-helical bundles. A module of three tandem spectrin repeats was used as a rigid building block self-assembling via coiled-coil (CC) dimer-forming peptides. CC peptides were precisely integrated to maintain the spectrin α-helix continuity in an appropriate frame to form extended nanorods. An orthogonal set of customizable CC heterodimers was harnessed for modular rigid domain association, which could be additionally regulated by metal ions and chelators. We achieved a robust assembly of rigid rods several micrometers in length, determined by atomic force microscopy and negative stain transmission electron microscopy. Furthermore, these rigid rods can serve as a scaffold for the decoration of diverse proteins or biologically active peptides along their length with adjustable spacing up to tens of nanometers, as confirmed by the DNA-PAINT super-resolution microscopy. This demonstrates the potential of modular bottom-up protein engineering and tunable CCs for the fabrication of functionalized protein biomaterials.

Avian deltacoronaviruses encode fusion-associated small transmembrane proteins that can induce syncytia formation

Fusion-associated small transmembrane (FAST) proteins are nonstructural viral proteins that induce cell-cell fusion. FAST proteins, which previously were identified in the genomes of double-stranded RNA viruses, typically contain an acylated N-terminal ectodomain, central transmembrane domain, and C-terminal endodomain with a polybasic region. Using sequence homology and protein motif prediction, we identified accessory proteins in a subset of avian deltacoronaviruses as putative FAST proteins. Transient expression of thrush coronavirus NS7b or common moorhen coronavirus NS7a, but not night heron coronavirus NS7b, induced cell-cell fusion. Syncytia were detected in primate kidney epithelial cells or fibroblasts but not chicken embryo fibroblasts, and addition of an N-terminal FLAG peptide to the proteins ablated fusion activity. These findings suggest that multiple avian deltacoronaviruses, positive-sense RNA viruses, encode nonstructural proteins that can mediate cell-cell fusion and share features with known FAST proteins. Additional studies are needed to understand contributions of these proteins to deltacoronavirus biology.

Structural and functional insights into the T-even type bacteriophage topoisomerase II.

T-even type bacteriophages are virulent phages commonly used as model organisms, playing a crucial role in understanding various biological processes. One such process involves the regulation of DNA topology during phage replication upon host infection, governed by type IIA DNA topoisomerases. In spite of various studies on prokaryotic and eukaryotic counterparts, viral topoisomerase II remains insufficiently understood, especially the unique domain composition of T4 phage. In this study, we determine the cryo-EM structures of topoisomerase II from T4 and T6 phages, including full-length structures of both apo and DNA-binding states which have never been determined before. Together with other conformational states, these structures provide an explicit blueprint of mechanisms of phage topoisomerase II. Particularly, the asymmetric dimeric interactions observed in cryo-EM structures of T6 phage topoisomerase II ATPase domain and central domain bound with DNA shed light on the asynchronous ATP usage and asynchronous cleavage of the G-segment DNA, respectively. The elucidation of phage topoisomerase II's structures and functions not only enhances our understanding of mechanisms and evolutionary parallels with prokaryotic and eukaryotic homologs but also highlights its potential as a model for developing type IIA topoisomerase inhibitors.

Structure of the Nipah virus polymerase phosphoprotein complex

The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen.

Molecular Basis of the Recognition of the Active Rab8a by Optineurin

Optineurin (OPTN), a multifunctional adaptor protein in mammals, plays critical roles in many cellular processes, such as vesicular trafficking and autophagy. Notably, mutations in optineurin are directly associated with many human diseases, such as amyotrophic lateral sclerosis (ALS). OPTN can specifically recognize Rab8a and the GTPase-activating protein TBC1D17, and facilitate the inactivation of Rab8a mediated by TBC1D17, but with poorly understood mechanism. Here, using biochemical and structural approaches, we systematically characterize the interaction between OPTN and Rab8a, revealing that OPTN selectively recognizes the GTP-bound active Rab8a through its leucine-zipper domain (LZD). The determined crystal structure of OPTN LZD in complex with the active Rab8a not only elucidates the detailed binding mechanism of OPTN with Rab8a but also uncovers a unique binding mode of Rab8a with its effectors. Furthermore, we demonstrate that the central coiled-coil domain of OPTN and the active Rab8a can simultaneously interact with the TBC domain of TBC1D17 to form a ternary complex. Finally, based on the OPTN LZD/Rab8a complex structure and relevant biochemical analyses, we also evaluate several known ALS-associated mutations found in the LZD of OPTN. Collectively, our findings provide mechanistic insights into the interaction of OPTN with Rab8a, expanding our understanding of the binding modes of Rab8a with its effectors and the potential etiology of diseases caused by OPTN mutations.

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5'-OH kinase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for anti-fungal drug discovery. Mucorales species, deemed high priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO4 and 5'-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity.

Structures of the Varicella Zoster Virus Glycoprotein E and Epitope Mapping of Vaccine-Elicited Antibodies.

Background: Varicella zoster virus (VZV) is the causative agent for chickenpox and herpes zoster (HZ, shingles). HZ is a debilitating disease affecting elderly and immunocompromised populations. Glycoprotein E (gE) is indispensable for viral replication and cell-to-cell spread and is the primary target for anti-VZV antibodies. Importantly, gE is the sole antigen in Shingrix, a highly efficacious, AS01B-adjuvanted vaccine approved in multiple countries for the prevention of HZ, yet the three-dimensional (3D) structure of gE remains elusive. Objectives: We sought to determine the structure of VZV gE and to understand in detail its interactions with neutralizing antibodies. Methods: We used X-ray crystallography and cryo-electron microscopy to elucidate structures of gE bound by recombinant Fabs of antibodies previously elicited through vaccination with Zostavax, a live, attenuated vaccine. Results: The 3D structures resolve distinct central and C-terminal antigenic domains, presenting an array of diverse conformational epitopes. The central domain has two beta-sheets and two alpha helices, including an IgG-like fold. The C-terminal domain exhibits 3 beta-sheets and an Ig-like fold and high structural similarity to HSV1 gE. Conclusions: gE from VZV-infected cells elicits a human antibody response with a preference for the gI binding domain of gE. These results yield insights to VZV gE structure and immunogenicity, provide a framework for future studies, and may guide the design of additional herpesvirus vaccine antigens. Teaser: Structures of varicella zoster virus glycoprotein E reveal distinct antigenic domains and define epitopes for vaccine-elicited human antibodies.

In Silico Structural Prediction for the Generation of Novel Performant Midi-Dystrophins Based on Intein-Mediated Dual AAV Approach.

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.

Molecular consequences of SCA5 mutations in the spectrin-repeat domains of β-III-spectrin.

Spinocerebellar ataxia type 5 (SCA5) mutations in the protein β-III-spectrin cluster to the N-terminal actin-binding domain (ABD) and the central spectrin-repeat domains (SRDs). We previously reported that a common molecular consequence of ABD-localized SCA5 mutations is increased actin binding. However, little is known about the molecular consequences of the SRD-localized mutations. It is known that the SRDs of β-spectrin proteins interact with α-spectrin to form an α/β-spectrin dimer. In addition, it is known that SRDs neighbouring the β-spectrin ABD enhance actin binding. Here, we tested the impact of the SRD-localized R480W and the E532_M544del mutations on the binding of β-III-spectrin to α-II-spectrin and actin. Using multiple experimental approaches, we show that both the R480W and E532_M544del mutants can bind α-II-spectrin. However, E532_M544del causes partial uncoupling of complementary SRDs in the α/β-spectrin dimer. Further, the R480W mutant forms large intracellular inclusions when co-expressed with α-II-spectrin in cells, supporting that R480W mutation grossly disrupts the α-II/β-III-spectrin physical complex. Moreover, actin-binding assays show that E532_M544del, but not R480W, increases β-III-spectrin actin binding. Altogether, these data support that SRD-localized mutations alter key interactions of β-III-spectrin with α-II-spectrin and actin.

Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits.

5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.