Xin Yuan | PubMed Health

PubMed2026

Seed-type vacuolar processing enzymes recognize the 619th asparagine residue to posttranslationally cleave the HMW-GS 1Dy10-m619SN allele.

High molecular weight glutenin subunits (HMW-GSs) are critical grain storage proteins in wheat, which govern its unique processing quality. A HMW-GS 1Dy10 allele variant (1Dy10-m619SN), carrying a serine-to-asparagine substitution at the 619th residue, undergoes partial posttranslational cleavage. This modification leads to improved cookie-making quality. However, the enzymes mediating this cleavage remain unknown. In this study, we identified vacuolar processing enzymes (VPEs) as candidates for 1Dy10-m619SN processing using TurboID-based proximity labeling and RNA-seq analysis. In vitro cleavage assays confirmed that VPEs catalyzed 1Dy10-m619SN cleavage. Phylogenic analysis revealed that there are two seed-type VPEs in wheat, TaVPEI and TaVPEII, with TaVPEI being further subdivided into TaVPEI-1, TaVPEI-2, and TaVPEI-3. Despite sharing conserved catalytic domains, these isoforms display distinct temporal expression patterns, with TaVPEI-1 expression showing the strongest correlation with the posttranslational cleavage of 1Dy10-m619SN. TaVPEI-1 protein is localized to the vacuole, the well-known deposition site for HMW-GSs. Overexpression of TaVPEI-1 in wheat enhances the 1Dy10-m619SN cleavage. Collectively, these findings demonstrate that the seed-type VPEs in wheat are responsible for the posttranslational cleavage of 1Dy10-m619SN, which provides new insights into the molecular basis of wheat's unique processing quality.

Triticum↗

PubMed2025

Novel mutations associated with clofazimine resistance in Mycobacterium intracellulare.

BACKGROUND: Clofazimine is a promising repurposed drug for treating Mycobacterium avium-intracellulare complex pulmonary disease, but its resistance mechanisms in Mycobacterium intracellulare remain poorly understood. OBJECTIVE: This study aims to elucidate the resistance mechanisms of M. intracellulare to clofazimine. METHODS: We isolated 36 clofazimine-resistant M. intracellulare mutants in vitro and performed whole-genome sequencing to identify resistance-associated mutations. Gene complementation was used to validate the role of the identified mutations. RESULTS: We identified various mutations in the marR gene (WP_009952290.1) in 61% of clofazimine-resistant mutants by whole-genome sequencing. Mutations were identified in additional genes encoding ssuD (flavin-dependent oxidoreductase, C67A), lppI (membrane lipoprotein, C207 deletion), GMC oxidoreductase (glucose-methanol-choline oxidoreductase, G157 deletion), MASE1 domain-containing protein (C62G) and PPE family protein (222C deletion). Gene complementation experiments demonstrated that introducing the wild-type marR in clofazimine-resistant strain (L72) with marR mutations reduced clofazimine MIC from 1 mg/L to susceptible baseline (0.25 mg/L), confirming its critical role in clofazimine resistance. Notably, the M. intracellulare MarR lacks homology to Mycobacterium tuberculosis MarR family protein Rv0678 (MmpR) involved in clofazimine and bedaquiline resistance but is flanked by non-efflux pump genes (dhmA and doxX), and unlike M. tuberculosis, its mutation does not cause bedaquiline cross-resistance, indicating a different MarR and distinct regulatory mechanism for clofazimine resistance in M. intracellulare. CONCLUSIONS: This work highlights marR as a key determinant of clofazimine resistance in M. intracellulare and underscores the need for further mechanistic studies with implications for rapid molecular detection and effective treatment.

Clofazimine↗