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Arsenic is the most common toxic metalloid in the environment. Nearly all organisms have genes for arsenic detoxification. Arsenic detoxification genes are frequently organized in chromosomal or plasmid-encoded arsenic resistance (ars) operons, which are commonly regulated by members of the ArsR transcriptional repressors. To date, three As(III)-responsive ArsRs with different As(III) binding sites have been identified. Here, we identify a new type of As(III)-responsive ArsR repressor that has an atypical As(III) binding site and controls transcription of the ars operon of Arsenicibacter rosenii SM-1. Our results provide new insights into the classification and evolution relationship of the ArsR transcriptional repressors.

Cytochrome c maturation (CCM), a posttranslational modification involving covalent attachment of heme to polypeptides (apocyt c), is essential for the activity and cellular function of cytochromes c. Here, we identify and substantiate CcmB as heme translocase in bacteria. When in excess, CcmB expels intracellular heme into the periplasm and thus is detrimental to the cell. We then show that complexation with CcmACD ensures heme translocated by CcmB to be used for CCM only. Moreover, structural analysis and atomistic molecular dynamics simulations reveal that CcmB absorbs heme from the membrane to a heme pocket formed in the dimer interface of the transmembrane helix-bundles. These data, collectively by providing detailed insights into the conformational landscape of CcmB during heme entry, fill in the missing link in our understanding of the heme translocation for CCM.

Glycosylphosphatidylinositol (GPI) anchoring is one of the conserved posttranslational modifications in eukaryotes that attach proteins to the plasma membrane. In fungi, in addition to plasma membrane GPI-anchored proteins (GPI-APs), some GPI-APs are specifically released from the cell membrane, secreted into the cell wall, and covalently linked to cell wall glucans as GPI-anchored cell wall proteins (GPI-CWPs). However, it remains unclear how fungal cells specifically release GPI-CWPs from their membranes. In this study, phospholipase PlcH was identified and confirmed as a phospholipase C that hydrolyzes phosphate ester bonds to release GPI-APs from the membrane of the opportunistic fungal pathogen Aspergillus fumigatus. Deletion of the plcH gene led to abnormal conidiation, polar abnormality, and increased sensitivity to antifungal drugs. In an immunocompromised mouse model, the ΔplcH mutant showed an attenuated inflammatory response and increased macrophage killing compared with the wild type. Biochemical and proteomic analyses revealed that PlcH was involved in the localization of various cell wall GPI-APs and contributed to the cell wall integrity. Our results demonstrate that PlcH can specifically recognize and release GPI-CWPs from the cell membrane, which represents a newly discovered secretory pathway of GPI-CWPs in A. fumigatus.

Optimizing enzyme thermostability is essential for advancements in protein science and industrial applications. Currently, (semi-)rational design and random mutagenesis methods can accurately identify single-point mutations that enhance enzyme thermostability. However, complex epistatic interactions often arise when multiple mutation sites are combined, leading to the complete inactivation of combinatorial mutants. As a result, constructing an optimized enzyme often requires repeated rounds of design to incrementally incorporate single mutation sites, which is highly time-consuming. In this study, we developed an AI-aided strategy for enzyme thermostability engineering that efficiently facilitates the recombination of beneficial single-point mutations. We utilized thermostability data from creatinase, including 18 single-point mutants, 22 double-point mutants, 21 triple-point mutants, and 12 quadruple-point mutants. Using these data as inputs, we used a temperature-guided protein language model, Pro-PRIME, to learn epistatic features and design combinatorial mutants. After two rounds of design, we obtained 50 combinatorial mutants with superior thermostability, achieving a success rate of 100%. The best mutant, 13M4, contained 13 mutation sites and maintained nearly full catalytic activity compared to the wild-type. It showed a 10.19°C increase in the melting temperature and an ~655-fold increase in the half-life at 58°C. Additionally, the model successfully captured epistasis in high-order combinatorial mutants, including sign epistasis (K351E) and synergistic epistasis (D17V/I149V). We elucidated the mechanism of long-range epistasis in detail using a dynamics cross-correlation matrix method. Our work provides an efficient framework for designing enzyme thermostability and studying high-order epistatic effects in protein-directed evolution.

Advances in deep learning have significantly aided protein engineering in addressing challenges in industrial production, healthcare, and environmental sustainability. This review frames frequently researched problems in protein understanding and engineering from the perspective of deep learning. It provides a thorough discussion of representation methods for protein sequences and structures, along with general encoding pipelines that support both pre-training and supervised learning tasks. We summarize state-of-the-art protein language models, geometric deep learning techniques, and the combination of distinct approaches to learning from multi-modal biological data. Additionally, we outline common downstream tasks and relevant benchmark datasets for training and evaluating deep learning models, focusing on satisfying the particular needs of protein engineering applications, such as identifying mutation sites and predicting properties for candidates' virtual screening. This review offers biologists the latest tools for assisting their engineering projects while providing a clear and comprehensive guide for computer scientists to develop more powerful solutions by standardizing problem formulation and consolidating data resources. Future research can foresee a deeper integration of the communities of biology and computer science, unleashing the full potential of deep learning in protein engineering and driving new scientific breakthroughs.

In silico computational methods have been widely utilized to study enzyme catalytic mechanisms and design enzyme performance, including molecular docking, molecular dynamics, quantum mechanics, and multiscale QM/MM approaches. However, the manual operation associated with these methods poses challenges for simulating enzymes and enzyme variants in a high-throughput manner. We developed the NAC4ED, a high-throughput enzyme mutagenesis computational platform based on the "near-attack conformation" design strategy for enzyme catalysis substrates. This platform circumvents the complex calculations involved in transition-state searching by representing enzyme catalytic mechanisms with parameters derived from near-attack conformations. NAC4ED enables the automated, high-throughput, and systematic computation of enzyme mutants, including protein model construction, complex structure acquisition, molecular dynamics simulation, and analysis of active conformation populations. Validation of the accuracy of NAC4ED demonstrated a prediction accuracy of 92.5% for 40 mutations, showing strong consistency between the computational predictions and experimental results. The time required for automated determination of a single enzyme mutant using NAC4ED is 1/764th of that needed for experimental methods. This has significantly enhanced the efficiency of predicting enzyme mutations, leading to revolutionary breakthroughs in improving the performance of high-throughput screening of enzyme variants. NAC4ED facilitates the efficient generation of a large amount of annotated data, providing high-quality data for statistical modeling and machine learning. NAC4ED is currently available at http://lujialab.org.cn/software/.

The aquifer in the subseafloor igneous basement is a massive, continuous microbial substrate, yet sparingly little is known about life in this habitat. The work to date has focused largely on describing microbial diversity in the young basement (<10 Ma), where the basaltic crust is still porous and fluid flow through it is active. Here, we test the hypothesis that microbial life exists in subseafloor basement >65 Ma using samples collected from the Louisville Seamount Chain via seafloor drilling. Cell biomass was heterogeneous in nature, ranging from below detection to ~10⁴ cells cm^-3. Bacterial 16S rRNA genes from core samples and enrichment incubations are dominated by lineages putatively carrying out nitrogen, sulfur, and metal redox processes and hydrocarbon oxidation. Taken together, the data indicate that microbial life is indeed present in subseafloor igneous basement >65 Ma, which significantly expands the range of the subseafloor biosphere where microbial life is known to exist.

Through the analysis of data from children aged 6 months to 8 years enrolled in the Vitamin D Antenatal Asthma Reduction Trial (VDAART), significant simultaneous associations were identified between variants in the fragile histidine triad (FHIT) gene, children's body mass index, microbiome features related to obesity, and key lipids and amino acids. These patterns represent evidence of the genotype influence in shaping the host microbiome in developing stages and new potential biomarkers for childhood obesity, insulin resistance, and type 2 diabetes.