Biology subjects

Hao, B.

Publications and source records attributed to Hao, B..

At least 19 recordsLinked to original sources

bioRxiv2026

An entropy-based diagnostic framework for characterizingmethylation state dynamics during preimplantation development

DNA methylation undergoes predictable changes with age, and preimplantation embryos are known to undergo global epigenetic reprogramming. However, the specific fate of age associated methylation signatures during early development has not been systematically quantified. Using published human sperm age associated differentially methylated regions (DMRs) as a feature space, we integrated single cell methylome and transcriptome data to develop the Transgenerational Reset Operator (TRO), a computational framework for profiling preimplantation stages. We found that the morula stage represents the nadir of age associated methylation entropy while retaining high developmental potency, distinguishing it from a simple demethylation endpoint. Dynamical modelling revealed that independent DMR drift fails to recapitulate the morula state, requiring a coordinated, structured correction concentrated in specific DMR subsets and modules with marked directional sensitivity. Independent chromatin accessibility data supported a stage specific methylation accessibility coupling at morula, albeit with modest effect sizes. Cross species mouse and orthogonal multiomic evidence suggested partial conservation but with weight dependence and heterogeneity. Collectively, our study defines morula as a computational "ground zero" candidate for age associated methylation features and proposes a testable hypothesis of developmental regulation, while emphasizing that matched parental offspring perturbation experiments are needed to establish causal mechanisms.

developmental biology↗

bioRxiv2026

Mechanisms of USP7/MAGEL2 Complex Assembly and Its Mutational Disruption in Neurodevelopmental Diseases

The WASH complex regulates endosomal trafficking and is linked to several neurodevelopmental diseases, including Prader-Willi syndrome, Schaaf-Yang syndrome, and Hao-Fountain syndrome. Its function is tightly controlled by ubiquitination, maintained by the multi-subunit MUST complex containing both a ubiquitin ligase (MAGEL2/TRIM27) and a deubiquitinase (USP7). However, the mechanism underlying the MUST complex assembly remains poorly understood. In this study, we investigate the assembly of USP7 and MAGEL2 components of the MUST complex using NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and cellular assays. We show that the USP7/MAGEL2 interaction is bipartite and multivalent. Two distinct domains of USP7, TRAF and UBL1-2, recognize two unstructured but evolutionarily conserved regions of MAGEL2, one of which contains multiple TRAF-binding sites. Furthermore, we determine the high-resolution crystal structure of the TRAF/MAGEL2 complex and identify Hao-Fountain syndrome-linked mutations in USP7 that disrupt USP7/MAGEL2 complex formation in vitro and in cells. These findings provide mechanistic insight into the pathogenic basis of Hao-Fountain syndrome and related Schaaf-Yang and Prader-Willi syndromes.

biochemistry↗

bioRxiv2026

PXL: a Nucleic Acid-Binding Module of Promyelocytic Leukemia Protein

The promyelocytic leukemia protein (PML) is a stress-response factor that assembles into PML nuclear bodies, dynamic subnuclear compartments involved in tumor suppression and antiviral defense. The most abundant isoform, PML-1, has been linked to transcriptional regulation, genome stability, and antiviral responses, yet the molecular basis of these functions remains unclear. Here, we report that PML-1 contains a unique nucleic acid- binding module, PXL, and determine its three-dimensional structure by X-ray crystallography. Further biochemical, mutational, and cellular analyses, including RNA-seq, demonstrate that this module selectively binds single-stranded G-rich RNA and DNA motifs and modulates the transcriptome. These findings reveal an unexpected molecular function of PML and provide a framework for understanding its roles in nuclear organization and gene regulation.

biochemistry↗

bioRxiv2026

Endothelial PTBP1 Deletion in Transplanted Cardiac Tissue Limits Cardiac Allograft Vasculopathy

BackgroundCardiac allograft vasculopathy (CAV) is a leading cause of late graft failure and mortality following heart transplantation, with limited therapeutic options. Endothelial cells (ECs), at the interface between the donor graft and host immune system, play a central role in CAV development. However, the molecular mechanisms driving endothelial dysfunction and vascular remodeling in chronic heart transplant rejection remain poorly understood. MethodsTo characterize endothelial alterations associated with CAV, we isolated nuclei from cardiac tissues of four human donor groups: (1) early post-transplant CAV-negative surveillance biopsies, (2) CAV-negative explanted grafts with acute cellular rejection (ACR), (3) late-stage CAV-positive explanted grafts, and (4) naive non-transplanted control hearts. We applied intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq) to profile endothelial gene expression together with nuclear protein levels of splice factor polypyrimidine tract-binding protein 1 (PTBP1), a key post-transcriptional regulator of endothelial inflammatory responses. Functional relevance of PTBP1 was assessed using endothelial-specific deletion of Ptbp1 in an F1 hybrid murine model of CAV. ResultsIn human CAV, endothelial cells exhibited increased transforming growth factor-{beta} (TGF-{beta}) signaling and reduced oxidative phosphorylation (OxPhos) transcripts. Nuclear PTBP1 protein levels were markedly elevated in CAV endothelium and were associated with TGF-{beta}-responsive transcriptional programs and correlated with clinical indices of cardiac dysfunction. In murine heart transplants, endothelial-specific deletion of Ptbp1 markedly reduced hallmarks of CAV, including neointimal hyperplasia, fibrosis, and lymphocyte activation. At the molecular level, endothelial Ptbp1 deletion prevented suppression of mitochondrial transcripts and preserved mitochondrial content and integrity under hypoxic stress, attenuating interferon signaling in endothelial cells. ConclusionThese findings identify PTBP1 as a central endothelial regulator linking pro-fibrotic stress to mitochondrial dysfunction and immune activation in chronic cardiac allograft rejection. Targeting endothelial PTBP1 may represent a strategy to limit chronic graft injury while minimizing systemic immunosuppression.

immunology↗

bioRxiv2025

SF3B1 phosphorylation is an evolutionarily conserved step in spliceosome activation carried out by the divergent, OTS964-insensitive kinase CRK9 in trypanosomes

SF3B1 is a subunit of the heptameric SF3B complex which, as part of the U2 small nuclear ribonucleoprotein, facilitates branch point recognition in pre-mRNA splicing. In addition to this early-stage function, it was recently shown that activation of the spliceosome depends on the phosphorylation of threonine-proline (TP) motifs in SF3B1s N-terminal domain (NTD) by cyclin-dependent kinase 11 (CDK11). This breakthrough result was made possible by the discovery of the CDK11-specific inhibitor OTS964. Trypanosomes are protistan parasites whose proteomes are highly divergent in sequence from those of model organisms, and thus their CDKs were generically named CDC2-related kinases (CRKs). We previously characterized the trimeric CRK9 complex of Trypanosoma brucei and showed that it is essential for spliced leader trans splicing, the predominant splicing mode in the parasite. Although CRK9 and CDK11 deviate from each other substantially, we show that CRK9 activity is required to maintain SF3B1 phosphorylation in vivo, CRK9 directly phosphorylates TP motifs in the SF3B1 NTD in vitro, and the TP motifs themselves are crucial for spliceosome activation, demonstrating evolutionary conservation of this essential splicing step. Contrary to CDK11 and human cells, CRK9 and trypanosomes were rather insensitive to OTS964, indicating potentially exploitable differences in their ATP-binding pockets.

molecular biology↗

bioRxiv2025

CDR-aware masked language models for pairedantibodies enable state-of-the-art bindingprediction

BackgroundTherapeutic antibodies are a leading class of biologics, yet their unique architecture poses challenges for computational modeling. Each antibody comprises paired heavy and light variable domains with conserved framework regions that maintain structure and hypervariable complementarity-determining regions (CDRs) that directly contact antigens. This functional asymmetry, where CDRs determine binding specificity while frameworks provide scaffolding, suggests that region-aware training strategies could yield superior representations. Existing protein language models treat all regions uniformly, potentially missing critical features present in CDRs. MethodsWe developed a region-aware pretraining strategy for paired variable domain sequences using two protein language models: a 3 billion parameter model (ESM2) and a compact 600 million parameter model (ESM C). We compared three masking approaches: uniform whole-chain masking, CDR-focused masking, and a hybrid strategy. Final models were trained on over 1.6 million paired antibody sequences and evaluated on binding affinity datasets with over 90,000 antibody variants across six antigens, including single-mutant panels and combinatorial libraries. ResultsHere we show that CDR-focused training produces embeddings with superior predictive performance for antibody-antigen binding. Our approach achieves up to 27% improvements in binding affinity prediction compared to benchmarked antibody models. Remarkably, training exclusively on paired sequences proves sufficient; pretraining on billions of unpaired sequences provides no measurable benefit. Our compact model matches or exceeds larger antibody-specific baselines. ConclusionsThese findings establish that prioritizing paired sequences with CDR-aware supervision over scale and complex training schemes achieves both computational efficiency and predictive accuracy, providing a practical framework for next generation antibody language models.

bioinformatics↗

bioRxiv2024

Blood cultures at baseline and during persistent candidemia contain populations of genetically diverse Candida albicans strains that may differ in echinocandin tolerance and virulence

It is unknown whether within-patient Candida albicans diversity is common during bloodstream infections (BSIs). We determined whole genome sequences of 10 C. albicans strains from blood cultures (BCs) in each of 4 patients. BCs in 3 patients contained mixed populations of strains that differed by large-scale genetic variants, including chromosome (Chr) 5 or 7 aneuploidy (n=2) and Chr1 loss of heterozygosity (n=1). Chr7 trisomy (Tri7) strains from patient MN were attenuated for hyphal and biofilm formation in vitro compared to euploid strains, due at least in part to NRG1 over-expression. Nevertheless, representative Tri7 strain M1 underwent filamentation during disseminated candidiasis (DC) in mice. M1 was more fit than euploid strain M2 during DC and mouse gastrointestinal colonization, and in blood ex vivo. M1 and M2 exhibited identical echinocandin minimum inhibitory concentrations, but M2 was more tolerant to micafungin in vitro. Furthermore, M2 was more competitive with M1 in mouse kidneys following micafungin treatment than it was in absence of micafungin. Tri7 strains represented 74% of patient MNs baseline BC population, but after 1d and 3d of echinocandin treatment, euploid strains were 93% and 98% of the BC population, respectively. Findings suggest that echinocandin tolerant, euploid strains were a subpopulation to more virulent Tri7 strains at baseline and then were selected upon echinocandin exposure. In conclusion, BCs in at least some patients are comprised of diverse C. albicans populations not recognized by the clinical lab, rather than single strains. Clinical relevance of C. albicans diversity and antifungal tolerance merits further investigation.

microbiology↗

bioRxiv2024

Nematode Extracellular Protein Interactome Expands Connections between Signaling Pathways

Multicellularity was accompanied by the emergence of new classes of cell surface and secreted proteins. The nematode C. elegans is a favorable model to study cell surface interactomes, given its well-defined and stereotyped cell types and intercellular contacts. Here we report our C. elegans extracellular interactome dataset, the largest yet for an invertebrate. Most of these interactions were unknown, despite recent datasets for flies and humans, as our collection contains a larger selection of protein families. We uncover new interactions for all four major axon guidance pathways, including ectodomain interactions between three of the pathways. We demonstrate that a protein family known to maintain axon locations are secreted receptors for insulins. We reveal novel interactions of cystine-knot proteins with putative signaling receptors, which may extend the study of neurotrophins and growth-factor-mediated functions to nematodes. Finally, our dataset provides insights into human disease mechanisms and how extracellular interactions may help establish connectomes.

biochemistry↗

bioRxiv2023

Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration

AbstractDespite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimers Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear {beta}-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.

neuroscience↗

bioRxiv2023

TRACTION FORCE AND MECHANOSENSING CAN BE FUNCTIONALLY DISTINGUISHED THROUGH THE USE OF SPECIFIC DOMAINS OF THE CALPAIN SMALL SUBUNIT

Cell migration is a fundamental process pertaining to many critical physiological events. The ability to form and release adhesion structures is necessary for cell migration. The Calpain family of cysteine proteases are known to target adhesion proteins as their substrates and modulate adhesion dynamics. The two best studied Calpains, Calpain 1 and Calpain 2 form catalytically active holoenzymes through heterodimerization with a common non-catalytic regulatory small subunit known as Calpain 4. In previous studies, we determined that calpains are important in the production of traction forces and in the sensing of localized mechanical stimulation from the external environment. We found that perturbation of either Calpain 1 or 2 had no effect on the generation of traction forces. However, traction forces were weak when Calpain 4 was silenced. On the other hand, silencing of Calpain 1, 2, or 4 resulted in deficient sensing of external mechanical stimuli. These results together suggest that Calpain 4 functions independent of the catalytic large subunits in the generation of traction forces but functions together with either catalytic subunit in sensing external mechanical stimuli. The small subunit Calpain 4 contains 268 a.a. and is composed of 2 domains, the N-terminal domain V and C-terminal domain VI. Domain VI is a calmodulinlike domain containing five consecutive EF-hand motifs, of which the fifth one heterodimerizes with a large subunit. Moreover, domain V contains the common sequence GTAMRILGGVI that suggests cell membrane interactions. Given these attributes of domain V and VI of Calpain 4, we speculated that an individual domain might provide the functional properties for either traction or sensing. Therefore, each domain was cloned and expressed individually in Capn4-/- cells and assayed for traction and sensing. Results revealed that over-expression of domain V was sufficient to rescue the traction forces defect in Capn4-/- cells while overexpression of domain VI did not rescue the traction force. Consistent with our hypothesis, overexpression of domain VI rescued the sensing defect in Capn4-/- cells while overexpression of domain V had no effect. These results suggest that individual domains of Calpain 4 do indeed function independently to regulate either traction force or the sensing of external stimuli. We speculate that membrane association of Calpain 4 is required for the regulation of traction force and its association with a catalytic subunit is necessary for mechanosensing.

cell biology↗

bioRxiv2023

Regulation of Traction Force through the Direct Binding of Basigin and Calpain 4

Traction force and mechanosensing (the ability to sense the mechanical attributes of the environment) are two key factors that enable a cell to modify its behavior during migration. Previously, it was determined that the calpain small subunit, calpain 4 (CapnS1), regulates the production of traction force independent of its proteolytic holoenzyme. A proteolytic enzyme is formed by calpain 4 binding to either of its catalytic partners, calpain 1 and 2. To further understand how calpain 4 regulates traction force, we used two-hybrid analysis to identify more components of the traction pathway. We discovered that basigin, an integral membrane protein and a documented inducer of matrix-metalloprotease (MMP), binds to calpain 4 in two-hybrid and pull-down assays. Traction force was deficient when basigin was silenced in MEF cells, and this deficiency was also reflected in the defect in substrate adhesion strength. Unlike Capn4-/- MEF cells, the cells deficient in basigin had normal mechanosensing abilities. Together, these results implicate basigin in the pathway in which calpain 4 regulates traction force independent of the catalytic large subunits.

cell biology↗

bioRxiv2023

A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Force in vivo

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the {Delta}{psi} component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology (NanoBiT) in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions, {Delta}pH, in addition to {Delta}{psi}, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

biochemistry↗

bioRxiv2022

A positive statistical benchmark to assess network agreement

As the current best practice, an experimental network dataset is validated by showing significant overlap with a gold standard network. Significance is assessed by comparison to a negative benchmark, often a randomized version of the same gold standard. While such analysis can reliably indicate the presence of signal, it is illsuited to assess how much signal there is. As an alternative, here we introduce a positive statistical benchmark corresponding to the best-case scenario, capturing the maximum possible overlap between two networks. Such a positive benchmark can be efficiently generated in a maximum entropy framework and opens the way to assess if the observed overlap is significantly different from the best-case scenario. In combination with the negative benchmark, we provide a normalized overlap score (Normlap). As an application, we compare molecular and functional networks, resulting in an agreement network of human as well as yeast network datasets. Although the number of shared interactions between most networks within the same organism is relatively small, we find that it is typically close to the best-case scenario. This paradox is resolved by the underlying degree inconsistency, meaning that highly connected hubs in one network often have small degrees in another, limiting the potential overlap. Furthermore, we illustrate how Normlap improves the quality assessment of experimental networks, fostering the creation of future high-quality networks.

bioinformatics↗

bioRxiv2022

Pathway elucidation of pharmaceutical cucurbitacin IIa in Hemsleya chinensis and high-level production of precursor cucurbitadienol in engineered Saccharomyces cerevisiae and Nicotiana benthamiana

Cucurbitacin IIa is a triterpene isolated exclusively from Hemsleya plants, which is non-steroidal anti-inflammatory drug that function as the main ingredient of Hemslecin capsules and Supplemental Tablets in China. In this study, the biosynthetic pathway of cucurbitacin IIa was elucidated by characterization of squalene epoxidases (HcSE1, HcSE2), cucurbitenol synthases (HcOSC6) and acyltransferases (HcAT1) in Hemsleya chinensis. Meanwhile, cycloartenol synthases (HcOSC1), isomultiflorenol synthases (HcOSC5) and {beta}-amyrin synthase (HcOSC2-4) involved in sterol and triterpenes biosynthesis were functionally illustrated. The high-level production of yeast the key cucurbitacin precursor, cucurbitadienol, was constructed to produce 296.37 mg/L cucurbitadienol and 722.99 mg/L total triterpenoid which is the highest yield cucurbitadienol from known engineered microbes. Moreover, production of cucurbitenol in transient expression of tobacco was employed to achieve 94.8 mg/g dry weight (dw) cucurbitenol from leaves. In this study, the key genes involved in cucurbitacin IIa biosynthesis were identified to facilitate its medical applications via biosynthetic strategy. Meanwhile, the high-level production of cucurbitadienol chassis yeast and tobacco transient expression offered a robust and sSupplemental Table substrate for pharmaceutical cucurbitacin production and screening platform for candidate genes involved in cucurbitacin biosynthesis.

synthetic biology↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗

bioRxiv2022

Improved Predictions Of MHC-Peptide Binding Using Protein Language Models

AO_SCPLOWBSTRACTC_SCPLOWMajor histocompatibility complex (MHC) molecules bind to peptides from exogenous antigens, and present them on the surface of cells, allowing the immune system (T cells) to detect them. Elucidating the process of this presentation is essential for regulation and potential manipulation of the cellular immune system [1]. Predicting whether a given peptide will bind to the MHC is an important step in the above process, motivating the introduction of many computational approaches. NetMHCPan [2], a pan-specific model predicting binding of peptides to any MHC molecule, is one of the most widely used methods which focuses on solving this binary classification problem using a shallow neural network. The successful results of AI methods, especially Natural Language Processing (NLP-based) pretrained models in various applications including protein structure determination, motivated us to explore their use in this problem as well. Specifically, we considered fine-tuning these large deep learning models using as dataset the peptide-MHC sequences. Using standard metrics in this area, and the same training and test sets, we show that our model outperforms NetMHCpan4.1 which has been shown to outperform all other earlier methods [2].

molecular biology↗

bioRxiv2021

Functional analysis of the polar amino acid in TatA transmembrane helix

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (pmf) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different pmf components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

biochemistry↗

bioRxiv2021

Hydrophobic mismatch effect is a key factor in protein transport on the Tat pathway

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

biochemistry↗