Lin Y et al. · Jul 1, 2026
Homooligomerisation is a prevalent and important process that many proteins undergo to form the quaternary structures required for biological function. However, determining oligomeric states and structures experimentally remains technically challenging and time-consuming for many proteins. Here, we show that the protein structure prediction tools AlphaFold2-Multimer and AlphaFold3 can be used to quickly and accurately predict oligomeric states and structures for a range of soluble and membrane proteins. Across over 4700 proteins, AlphaFold2-Multimer provides reliable oligomeric state predictions in the majority of cases, however accuracy is more limited for proteins lacking close structural representatives in the AlphaFold training set, highlighting the dependence of these methods on robust training data. Together, our results suggest both the utility and current limitations of AlphaFold-based oligomeric state prediction, highlight cases where multiple physiologically relevant assemblies may be plausible, and provide practical guidance for minimizing computational cost, identifying challenging cases, and applying these methods to proteins lacking experimental structural data.
Biochemistry, Genetics and Molecular Biology
Parchuri P et al. · Jul 1, 2026
Triacylglycerols are the major energy storage lipids in plants, animals, and microorganisms, and are predominantly produced by acyl-CoA:diacylglycerol (DAG) acyltransferases (DGATs). Two enantiomers of the DAG substrate, sn-1,2 and sn-2,3, can be produced by different biological mechanisms; however, little is known about which species produce each enantiomer, the selectivity of DGAT isoforms for either enantiomer, or whether DGAT enantiomer selectivity varies across organisms. Here, DAG enantiomer selectivity of DGAT1 and DGAT2 was measured from eight seed plants, two mammals, one oleaginous yeast, and one photosynthetic microalga using enantiomer-specific in vitro DGAT assays. Across most plants, DGAT1 favored sn-1,2-DAG, whereas DGAT2 preferentially utilized sn-2,3-DAG. However, there were several exceptions. Mammalian DGAT1, DGAT2, and microbial DGAT1s efficiently used both DAG enantiomers, while microbial DGAT2s had unique selectivity. The selectivity of several DGATs for combined acyl-CoA and DAG enantiomer molecular species were also evaluated for biotechnical applications. Therefore, DGAT DAG enantiomer selectivity is common yet strongly dependent on lineage and isoform and likely shaped in part by species-specific metabolic context of triacylglycerol synthesis, turnover, and remodeling. This work expands our understanding of DGAT function and establishes a foundation for leveraging enantiomer-selective acyltransferases in metabolic engineering of tailored lipid products.
Biochemistry, Genetics and Molecular Biology
Narayan OP et al. · Jul 1, 2026
While recent research shows that biomolecular condensates play important roles in normal cellular processes and diseases, the driving forces in condensate formation are not well understood, especially regarding the role of structured self-associative protein domains. In this work, we study the contribution of a model structured domain, coiled-coil domain, in promoting condensate formation of fusion proteins (FPs). Starting from a large set of ~50,000 FPs, we systematically narrowed down to investigate 14 FPs and their corresponding 18 coiled-coil domains. We showed that all 14 FPs are capable of assembling condensates with high potency. When isolated from the rest of the protein contexts, 11 of the 18 coiled-coil domains can induce condensation on their own, despite their short length compared to their full-length counterparts. To understand the differences between coiled-coils that can drive condensate formation and those that cannot, we developed a "triad-extension" model and found the condensate-sufficient coiled-coil domains have a higher propensity to extend beyond perfectly end-to-end matched dimer/oligomer to promote condensate formation.
Biochemistry, Genetics and Molecular Biology
Huang X et al. · Jul 1, 2026
Aging is a multifactorial process driven by interconnected hallmarks, including chronic inflammation, mitochondrial dysfunction, genomic and epigenetic alterations, and dysregulated intercellular communication. Extracellular vesicles (EVs), naturally derived nanoscale membrane vesicles capable of transporting diverse bioactive cargoes across tissues and biological barriers, have emerged as a highly promising platform for regenerative and anti-aging therapeutics. In this review, we systematically summarize the multifaceted anti-aging mechanisms of EVs, including suppression of the senescence-associated secretory phenotype (SASP), remodeling of the immune microenvironment, mitochondrial restoration and metabolic reprogramming, DNA damage repair, epigenetic modulation, recovery of proteostasis, activation of regenerative signaling pathways, and cross-organ communication-mediated rejuvenation. Beyond mechanistic insights, we integrate the targeting biology and cellular entry properties of EVs, encompassing natural tropism determinants, engineered targeting strategies, biodistribution profiles, receptor-ligand interactions, intracellular trafficking, and subcellular cargo release. Unlike previous reviews focusing on a single EV source or isolated pathways, we establish a comprehensive framework connecting molecular mechanisms with delivery engineering, tissue targeting, biosafety assessment, scalable manufacturing, and clinical translation. We address major technical bottlenecks limiting EV therapeutics-including EV heterogeneity, suboptimal delivery efficiency, endosomal degradation, and the lack of standardized quality-control frameworks-while highlighting emerging solutions such as bioengineered EVs, hybrid vesicle platforms, biomaterial-assisted delivery systems, and ultrasound-enhanced targeting technologies. By bridging fundamental biology, nanomedicine engineering, and clinical translation, this review provides a strategic roadmap for the development of next-generation precision anti-aging nanotherapeutics with systemic regulatory capacity, translational feasibility, and broad clinical potential.
Biochemistry, Genetics and Molecular Biology
Wang Y et al. · Jul 1, 2026
Sema3c is specifically expressed in the cardiac outflow tract (OFT) of the developing mouse heart and has been implicated in OFT polarization and great artery formation. However, the regulatory basis underlying its spatially restricted expression remains unclear. To investigate the mechanisms underlying OFT-specific Sema3c expression, we utilized chromatin accessibility data from distinct segments of the developing heart and identified a differentially accessible region as an OFT-specific Sema3c enhancer candidate. Unlike previously characterized Sema3c enhancers, this region is located distal to the transcription start site. Reporter analysis using transgenic mouse embryos demonstrated that this region exhibits transcriptional activity from E8.5 onward and remains specifically active in the OFT myocardium throughout heart development. We further defined a minimal 603 bp enhancer whose activity depends on GATA binding sites. This enhancer provides insight into the mechanisms underlying spatially restricted Sema3c expression involved in OFT development.
Biochemistry, Genetics and Molecular Biology
Kvasov NA et al. · Jul 1, 2026
Mitochondria import the majority of their proteins from the cytosol, creating a fundamental challenge: precursor proteins must be synthesized, maintained in an import-competent state, and delivered to mitochondrial translocases without premature folding or aggregation. While mitochondrial protein import has been considered a post-translational process, growing evidence shows that a subset of mitochondrial proteins is synthesized in proximity to the organelle. We term this process co-translational targeting, or local translation. It may lead to direct structural coupling of protein synthesis and import, which we term co-translational translocation. New approaches, including selective ribosome profiling, proximity labeling, and RNA imaging, reveal that mitochondrial mRNA localization is highly dynamic and can be driven by both RNA-based and translation-dependent mechanisms. In contrast to the well-defined signal recognition particle pathway at the endoplasmic reticulum, mitochondrial targeting appears to rely on more flexible mechanisms shaped by nascent-chain properties, translation elongation, and coding-sequence features beyond the targeting signal. We discuss how these processes may support mitochondrial biogenesis and proteostasis while also creating vulnerabilities associated with ribosome stalling and precursor quality control. Together, recent findings position mitochondrial protein targeting as an integral part of cellular protein biogenesis and highlight key open questions in the coordination of translation and organelle function.
Biochemistry, Genetics and Molecular Biology
Das R et al. · Jul 1, 2026
CYP2J2 is a membrane-bound cytochrome P450 that is expressed in cardiomyocytes, where it is known to metabolize arachidonic acid into cardioprotective epoxyeicosatrienoic acids (EETs). It consists of transmembrane domains embedded in the hydrophobic segments of the cell membrane and surrounded by various lipids. Currently, we lack a detailed understanding of the role of specific lipids in mediating the physicochemical properties of CYP2J2 and the factors that govern this phenomenon. In this study, CYP2J2 was reconstituted into nanodiscs with different lipid compositions, selected to reflect those of the endoplasmic reticulum (ER) membrane, where the enzyme is expressed. Using a combination of Nano-Differential Scanning Fluorimetry (nano-DSF) and UV-Visible Spectroscopy, we demonstrate that CYP2J2 undergoes a transition in its unfolding behavior between the detergent micelle and the nanodisc environment, with the first melting transition corresponding to heme perturbation. Furthermore, we show that altering the lipid environment causes shifts of up to 3-4°C and 8-9°C in the first and second melting transition temperatures, respectively, with sphingomyelin- and POPS (1-palmitoyl-2-oleoyl-glycero-3-phosphoserine) containing nanodiscs exhibiting the highest and lowest thermal stabilities, respectively. Lipid composition was found to have no effect on substrate (ebastine) binding affinities. However, NADPH-oxidation rates showed that lipid composition directly affects CYP2J2 function in nanodiscs by altering the rate of electron transfer between the CYP and its redox partner, Cytochrome P450 Reductase (CPR). Fluorescence anisotropy measurements with DPH (1,6-Diphenyl-1,3,5-hexatriene) were also used to characterize the membrane fluidity of cholesterol- and sphingomyelin-containing nanodiscs. Together, the results show that lipid composition directly modulates the thermal stability and functional properties of CYP2J2 in nanodiscs and underscore the importance of the charge of the lipid headgroup and membrane fluidity in our understanding of the mechanism by which lipid composition exerts these effects.
Biochemistry, Genetics and Molecular Biology
Molina OJ et al. · Jul 1, 2026
Modification of lysine residues is a common strategy in protein engineering, whether to prevent posttranslational modifications, control bioconjugation, or improve crystallization. The standard genetic approach-replacement with arginine by site-directed mutagenesis-preserves positive charge but alters other physicochemical attributes and cannot address the N-terminal amino group. Here, we characterize reductive methylation as a chemical alternative. This reaction converts every primary amino group to a dimethylamino group rapidly under mild aqueous conditions. Using human ribonuclease 1 and a cytotoxic variant engineered to evade the endogenous ribonuclease inhibitor as model systems, we assess the effects of complete dimethylation on thermostability, enzymatic catalysis, protein-protein interaction, compatibility with bioconjugation, cellular uptake, and intracellular persistence. Dimethylation preserves thermostability and a protein-protein interaction. Enzymatic catalysis, in contrast, is reduced by 10 2 - to 10 3 -fold, consistent with the role of catalytic lysine residues. Dimethylation is fully compatible with bioconjugation chemistry. Dimethylated and unmodified ribonucleases show comparable uptake and persistence in human cells. These findings establish reductive methylation as a practical and conservative strategy for lysine modification in protein and peptide engineering and support its use in applications such as biological proteolysis-targeting chimeras (bioPROTACs).
Biochemistry, Genetics and Molecular Biology
Li C et al. · Jul 1, 2026
Diabetic nephropathy (DN), a major complication of diabetes mellitus (DM), is characterized by severe clinical manifestations, impaired quality of life, and a high risk of progression to end-stage renal disease, underscoring the urgent need for effective therapeutic interventions. Mesenchymal stromal cell-derived exosomes (MSC-Exo) have emerged as promising candidates for mitigating inflammatory injury in DN due to their immunomodulatory properties, and exosomes derived from MSCs pretreated with inflammatory factors such as TNF-α and IFN-γ may possess enhanced therapeutic potential. In this study, exosomes isolated from human umbilical cord MSCs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Their therapeutic effects were evaluated in diabetic mice, focusing on renal inflammation and macrophage polarization. Both normal MSC-Exo (Norm-Exo) and TNF-α&IFN-γ-pretreated MSC-Exo (TNF-α&IFN-γ-Exo) effectively ameliorated kidney injury and promoted M2 macrophage polarization, with TNF-α&IFN-γ-Exo showing superior efficacy. High-glucose-stimulated RAW264.7 cells were used to explore the underlying mechanisms, and high-throughput RNA sequencing identified inhibitor of DNA binding 3 (ID3) as a molecule involved in MSC-Exo-regulated macrophage polarization. Loss-of-function experiments confirmed that ID3 knockdown alone recapitulated the effects of exosomes, promoting M2 polarization and suppressing M1 markers. Conversely, ID3 overexpression attenuated exosome efficacy. Mechanistically, ID3 partially mediated exosome-induced inhibition of the NF-κB pathway. The translational relevance of these findings was further validated in PMA-differentiated THP-1 human macrophages. Collectively, these findings demonstrate that MSC-Exo-particularly TNF-α&IFN-γ-Exo-attenuate diabetic renal injury by modulating macrophage polarization through ID3 regulation, highlighting a novel cell-free immunomodulatory approach for DN therapy.
Biochemistry, Genetics and Molecular Biology
Malkani R et al. · Jul 1, 2026
Objectives Ethical decision-making in veterinary practice and in animal welfare sectors can be particularly challenging. Decision-making is reported to be largely subjective with various influences biasing these decisions. To enhance the objectivity with ethical dilemmas and difficult decisions, the application of standardised tools is increasingly advocated. This study explores the utility of the Animal Welfare Assessment Grid (AWAG) as a decision-making tool in various sectors of dog welfare including veterinary medicine, shelter environments, and assistance dog organisations. Methods This was undertaken through an online mixed-methods questionnaire of clinicians using the AWAG in their workplace to assess dogs. A total of 38 respondents from a group of 99 veterinary and animal welfare professionals provided insights on the tool's functionality. Results Over 96% of respondents acknowledge the utility of the AWAG in aiding in decision-making and it is reported to facilitate discussions among colleagues and dog caregivers about welfare. While the overall sentiment towards the AWAG was predominantly positive, a minority of users expressed difficulties in using the tool, indicating areas where the tool could be further refined and improved. Clinical significance The results suggest that the AWAG serves as a valuable asset for veterinary clinicians and animal welfare professionals, aiding in the assessment of dogs' welfare and informing treatment and management decisions. By providing an instrument to aid in objective reasoning to the complex process of decision-making, this may help transform how welfare considerations are integrated into daily practice.
Biochemistry, Genetics and Molecular Biology