In silico characterization of a metatranscriptome-derived stress-responsive putative DnaJ/HSP40-domain-containing fragment associated with heavy metal homeostasis

Abstract

Heavy metal (HM)-contaminated soils impose severe stress on the environment, yet certain soil microbes exhibit remarkable tolerance, revealing adaptive mechanisms in stress-adapted communities. Micro-eukaryotic communities in such environments exhibit substantial transcriptional reprogramming to mitigate stress-induced cellular damage. This study explores the molecular basis of HM stress adaptation based on transcript-level evidence using RNA-Seq-based metatranscriptomics combined with comprehensive in silico characterization. Exp_525919, a highly expressed transcript, was annotated against the UniProt proteome database using BLAST and was predicted as an uncharacterized protein from Achlya hypogyna (ACHHYP_04605), showing 57% sequence identity and a significant e-value (7.6e-24). Integrative analyses, including functional annotation, structural modeling, domain architecture, and evolutionary conservation, suggest that Exp_525919 is a putative DnaJ/heat shock protein 40 (HSP40)-domain-containing fragment, potentially associated with cellular protein quality control processes under HM stress conditions. Secondary structure revealed a predominance of coil regions with interspersed β-strands and a central α-helix. GO annotation predicted potential associations with transport, nucleic acid binding, and ribosomal association. Structural modeling generated a high-confidence model (96% coverage, 99.5% confidence), consistent with predicted chaperone-like proteins. Protein–protein interaction analysis revealed that the predicted uncharacterized protein ACHHYP_04605 is closely associated with major molecular chaperones, including HSP70, DnaJ/HSP40, and HSP90, suggesting its involvement in HSP70-centered stress-responsive networks. Network clustering revealed a molecular chaperone architecture in which core protein folding components were identified as central network hubs potentially associated with stress-adaptive responses. Overall, RNA-Seq-based metatranscriptomic analysis identified transcriptionally responsive potential genes and pathways underlying HM tolerance in micro-eukaryotic communities and provided a robust computational foundation for future experimental validation.