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bioRxiv2025

Epistasis at the cell surface: what is the role of Erg3 loss-of-function in acquired echinocandin resistance?

Echinocandins, which target the fungal {beta}-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus (Candida glabrata) in which combined mutations in ERG3 and FKS2, but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude ERG3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic crosstalk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes.

molecular biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗

bioRxiv2018

Evaluation of the antifibrotic potency by knocking down SPARC, CCR2 and SMAD3

The genes of SPARC, CCR2, and SMAD3 are implicated in orchestrating inflammation and fibrosis in scleroderma and other fibrotic disorders. Aim of the studies was to examine synergistic effect of inhibition of these genes in treating fibrosis. The peptide nanoparticles were used to deliver the siRNAs in bleomycin-induced fibrotic mice. Triple combination of siRNAs targeting on Sparc, Ccr2 and Smad3 achieved favorable anti-inflammatory and anti-fibrotic effects. Inhibition of inflammation was evidenced by reduced inflammatory cells and proinflammatory cytokines in the BALF and/or the tissues. Activation of fibroblasts was suppressed in mouse tissues in which -Sma and collagens were significantly reduced. Aberrant expression of the genes in fibroblasts, monocytes/macrophage, endothelial and epithelial cells were reinstalled after the treatment. In addition, transcriptome profiles indicated that some bleomycin-induced alterations of multiple biological pathways were recovered to varying degrees by the treatment. The results indicated that the triple combination of siRNAs systemically reinstated multiple biopathways, probably through controlling on different cell types including fibroblasts, monocytes/macrophages, endothelial cells and others. The multi-target-combined therapeutic approach examined herein may represent a novel and effective therapy for fibrosis.

molecular biology↗

bioRxiv2022

The Rho GTPase exchange factor Vav2 promotes extensive age-dependent rewiring of the skin stem cell transcriptome

Both the number and regenerative activity of skin stem cells (SSCs) are regulated by Vav2, a GDP/GTP exchange factor involved in the catalytic stimulation of the GTPases Rac1 and RhoA. However, whether Vav2 signaling changes in SSCs over the mouse lifespan is not yet known. Using a mouse knock-in mouse model, we now show that the expression of a catalytically-active version of Vav2 (Vav2Onc) promotes an extensive rewiring of the overall transcriptome of SSCs, the generation of new transcription factor hubs, and the synchronization of many transcriptional programs associated with specific SSC states and well-defined signaling pathways. Interestingly, this transcriptome rewiring is not fixed in time, as it involves the induction of 15 gene expression waves with diverse distribution patterns during the life of the animals. These expression waves are consistent with the promotion by Vav2Onc of several functional SSC states that differ from those normally observed in wild-type SSCs. These results further underscore the role of Vav2 in the regulation of the functional state of SSCs. They also indicate that, unlike other Vav2-dependent biological processes, the signaling output of this exchange factor is highly contingent on age-dependent intrinsic and/or extrinsic SSC factors that shape the final biological readouts triggered in this cell type. AUTHOR SUMMARYSkin stem cells (SSCs) are essential for the homeostatic balance of the skin, yet little is known to date about the biological and molecular mechanisms that modulate their abundance, long-term stability, or functional states during ageing. To address this issue, in this work we have used a genetically-engineered gain-of-function mouse model for Vav2, a Rho guanine nucleotide exchange factor (GEF) that has been recently shown to be involved in skin stem cell homeostasis. By performing time-course genome-wide expression analyses combined with a number of computational methods, here we show that: (i) Vav2 plays a critical role in regulating the functional state of SSCs, and (ii) the signaling output of constitutively active Vav2 is highly contingent on age-dependent intrinsic and/or extrinsic SSC factors that shape the final biological readouts triggered in this cell type. We believe that these data represent, to our knowledge, one of the first examples of the time-dependent output of an oncogenic version of a Rho GEF along a wide time interval in mice.

cell biology↗

bioRxiv2020

ULK1 and ULK2 are less redundant than previously thought: Computational analysis uncovers distinct regulation and functions of these autophagy induction proteins

Macroautophagy, the degradation of cytoplasmic content by lysosomal fusion, is an evolutionary conserved process promoting homeostasis and intracellular defence. Macroautophagy is initiated primarily by a complex containing ULK1 or ULK2 (two paralogs of the yeast Atg1 protein). Deletion of ULK1 is sufficient to interrupt autophagy, while ULK2 seems expendable. To understand the differences between ULK1 and ULK2, we compared the human ULK1 and ULK2 proteins and their regulation. Despite the high similarity in their enzymatic domain, we found that ULK1 and ULK2 have major differences in their post-translational and transcriptional regulators. We identified 18 ULK1-specific and 7 ULK2-specific protein motifs serving as different interaction interfaces. We identified three ULK1-specific and one ULK2-specific transcription factor binding sites, and eight sites shared by the regulatory region of both genes. Importantly, we found that both their post-translational and transcriptional regulators are involved in distinct biological processes - suggesting separate functions for ULK1 and ULK2. For example, we found a condition-specific, opposite effect on apoptosis regulation for the two ULK proteins. Given the importance of autophagy in diseases such as inflammatory bowel disease and cancer, unravelling differences between ULK1 and ULK2 could lead to a better understanding of how autophagy is dysregulated in diseased conditions.

molecular biology↗

bioRxiv2020

Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase

Plasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo, and restriction endonuclease treatments.

molecular biology↗

bioRxiv2023

Using double cut in vitro assembled CRISPR/Cas9 to modify the genome of Coccidioides posadasii

Although the genus Coccidioides is divided into two closely related and putatively allopatric species, analysis shows that hybridization has occurred between species and at least one C. posadasii conserved fragment has introgressed into several C. immitis genomes in a population-specific manner. Transcript abundance in vitro and in vivo for ten ORFs in this introgressed region were measured for several isolates. We used signals of introgression and high mRNA transcript levels in the spherule as indicators of selection for genes related to critical biological processes involved in Coccidioides pathogenesis. The only transcript in the introgression region with significant expression was a gene that encodes for a betadefensin-like (DEFBL) peptide rich in serines and cysteines. Few virulence factors have been identified in Coccidioides, and we employed the CRISPR-Cas9 mediated gene deletion tool to delete this gene in Coccidioides.

molecular biology↗

bioRxiv2024

Development of immobilized antibody-based affinity grid strategy and application in on-grid purification of target proteins for high-resolution cryo-EM

In cryo-electron microscopy (cryo-EM), sample preparation, especially for rare or fragile macromolecular assemblies and those suffering from air-water interface denaturation and particle orientation distribution problems, is a major bottleneck. Here, we developed and characterized an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We used Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon covered grid surface. We showed that the IAAG grid can enrich the PA-tagged target proteins, and overcome preferred orientation problems. Furthermore, we demonstrated that our IAAG strategy can be utilized for on-grid purification of low-abundance target complexes from cell lysates and enables atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification feasible for high-resolution cryo-EM. This approach also has the potential for broader applicability in cryo-EM and cryo-ET.

molecular biology↗

bioRxiv2024

Dual modes of DNA N6-methyladenine maintenance by distinct methyltransferase complexes

Stable inheritance of DNA N6-methyladenine (6mA) is crucial for its biological functions in eukaryotes. Here, we identify two distinct methyltransferase (MTase) complexes, both sharing the catalytic subunit AMT1, but featuring AMT6 and AMT7 as their unique components, respectively. While the two complexes are jointly responsible for 6mA maintenance methylation, they exhibit distinct enzymology, DNA/chromatin affinity, genomic distribution, and knockout phenotypes. AMT7 complex, featuring high MTase activity and processivity, is connected to transcription-associated epigenetic marks, including H2A.Z and H3K4me3, and is required for the bulk of maintenance methylation. In contrast, AMT6 complex, with reduced activity and processivity, is recruited to initiate maintenance methylation immediately after DNA replication. These two complexes coordinate in maintenance methylation. By integrating signals from both replication and transcription, this mechanism ensures the faithful and efficient transmission of 6mA as an epigenetic mark in eukaryotes. Significance statementDNA N6-methyladenine (6mA) has recently been recognized as an epigenetic mark in eukaryotes. The stable inheritance of 6mA is essential for its biological functions. However, the precise mechanisms by which 6mA patterns are faithfully and efficiently transmitted remain largely unknown. Here, we have identified two distinct 6mA methyltransferase (MTase) complexes and elucidated their coordinated role in maintenance methylation. This dual- complex mechanism ensures rapid and accurate methylation at newly replicated loci with proper transcription-associated epigenetic marks.

molecular biology↗

bioRxiv2018

Evolutionary analysis of base-pairing interactions in DNA and RNA secondary structures

Pairs of nucleotides within biologically functional nucleic acid secondary structures often exhibit evidence of coevolution that is consistent with the maintenance of canonical base-pairing. MESSI is a sequence evolution model that infers substitution rates associated with base-paired sites in alignments of DNA or RNA sequences. MESSI can estimate these whilst simultaneously accounting for the uncertainty associated with an unknown RNA or DNA secondary structure shared across an alignment of sequences. Moreover, the unknown structure can be predicted, or a base-pairing probability matrix calculated. MESSI optionally leverages CUDA GPU parallelism to accelerate inference. MESSI was used to infer coevolution rates associated with GC, AU (AT in DNA), GU (GT in DNA) pairs in non-coding RNA alignments, and single-stranded RNA and DNA virus alignments. Inferred rates of GU pair coevolution were found to be higher at base-paired sites in single-stranded RNA viruses and non-coding RNAs than those of GT pairs in single-stranded DNA viruses, suggesting that GT pairs do not stabilise DNA secondary structures to the same extent as GU pairs in RNA. The relative coevolution rates associated with GC, AU, and GU pairs were largely consistent with their relative chemical base-pairing stabilities (GC base-pairs being more stable than AU base-pairs, and AU base-pairs being more stable than GU base-pairs). Additionally, MESSI estimates the degrees of coevolution at individual base-paired sites in an alignment. These estimates were computed for a SHAPE-MaP-determined HIV-1 NL4-3 RNA secondary structure and two corresponding alignments. MESSIs estimates of coevolution were significantly more strongly correlated with experimentally-determined SHAPE-MaP pairing scores as compared to three non-evolutionary measures of base-pairing covariation. Finally, to assist researchers in prioritising substructures with potential biological functionality, MESSI automatically identifies substructures and ranks them by degrees of coevolution at base-paired sites within them. Such a ranking was created for an HIV-1 subtype B alignment, revealing an excess of top-ranking substructures that have been previously identified in the literature as having structure-related functional importance, and a number of top-ranking structures that have not yet been characterised.

molecular biology↗

bioRxiv2019

Rad9/53BP1 promotes crossover recombination DNA repair by limiting the Sgs1 and Mph1 helicases

A DNA double strand break (DSB) is primed for homologous recombination (HR) repair through the nucleolytic processing (resection) of its ends, leading to the formation of a 3' single-stranded DNA (ssDNA). Generation of the ssDNA is accompanied by the loading of several repair factors, including the ssDNA binding factor RPA and the recombinase Rad51. Then, depending upon the availability and location of a homologous sequence, different types of HR mechanisms can occur. Inefficient or slow HR repair results in the activation of the DNA damage checkpoint (DDC)1. In budding yeast, the 53BP1 ortholog Rad9 acts as a scaffold, mediating signal from upstream kinases Mec1 and Tel1 (ATR and ATM in human) to downstream effectors kinases Rad53 and Chk1 (CHK2 and CHK1 in human). In addition to its role in DDC, Rad9 limits DSB resection 2. Remarkably, this function is conserved in 53BP1, also being implicated in cancer biology in human cells 3,4.\n\nHere we show that Rad9 limits the recruitment of the helicases Sgs1 and Mph1 on to a DSB, promoting Rad51-dependent recombination with long track DNA conversions, crossovers and break-induced replication (BIR). This regulation couples the DDC with the choice and effectiveness of HR sub-pathways, and might be critical to limit genome instability with implication for cancer research.

molecular biology↗

bioRxiv2019

Analysis of the expression of PIWI-interacting RNAs during cardiac differentiation of human pluripotent stem cells

PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted almost exclusively to germ line cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in somatic cells like pluripotent, neural, cardiac and even cancer cells. However, controversy still remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent stem cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNAs, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences for secondary piRNAs (ping-pong loop) were found. Genes hosting piRNAs in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that somatic piRNAs might have a role in fine-tuning the expression of genes involved in the differentiation of pluripotent cells to cardiomyocytes.

molecular biology↗

bioRxiv2026

Fluorescently guided workflow with rationally engineered 5' ligation adapters for high-sensitivity and low-bias small RNA sequencing

MicroRNAs (miRNAs) act as key regulators of gene expression across diverse cellular processes, and their precise quantification can provide unique insight into disease pathogenesis. High-throughput sequencing allows for comprehensive small RNA profiling; however, standard commercial library preparation workflows are challenged by issues of low sensitivity and representational bias, limiting reliable profiling, especially in scenarios where samples are scarce. Several structural studies have shown that this bias primarily arises due to sequence and secondary structure variations between miRNAs and adapters during enzyme-catalyzed biochemical reactions. In this work, we propose a new approach to ligation adapter engineering using a bioinformatic analysis of the human miRNome to rationally design structure-forcing 5 adapters, that physically override localized, unpredictable structural variations during the intermediate ligation state. We show that this approach combined with a practical fluorescence-guided workflow, utilizing a fluorescently-labeled 3 adapter and novel Fluorescent Ligation Rulers (FLRs) to guide precise band excision, can minimize representational bias and increase the sensitivity of small RNA sequencing from low-input biological matrices. In comprehensive benchmarks using a synthetic panel, this method significantly reduced bias and outperformed alternative commercial protocols. Finally, we demonstrate that this workflow enhances biomarker detection and library quality in challenging clinical matrices, especially in cerebrospinal fluid. Overall, this protocol enables highly accurate miRNome characterization and is well-suited for biomarker discovery in challenging sample types.

molecular biology↗

bioRxiv2021

TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease (CAD). ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating CAD; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EMEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease related biological substrates and facilitate activity-based ADAMTS7 biomarker development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/469331v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1520ad4org.highwire.dtl.DTLVardef@144f148org.highwire.dtl.DTLVardef@13cdbc1org.highwire.dtl.DTLVardef@7f7b4f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2023

GRable version 1.0: A software tool for site-specific glycoform analysis using the improved Glyco-RIDGE method with parallel clustering and MS2 information

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we have developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points are: "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large scale and in depth. Therefore, this software will help to analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can run freely online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/564073v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@12ae798org.highwire.dtl.DTLVardef@1cac352org.highwire.dtl.DTLVardef@dd6b92org.highwire.dtl.DTLVardef@c16ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

A comprehensive Schizosaccharomyces pombe atlas of physical transcription factor interactions with proteins and chromatin

Transcription factors (TFs) are key regulators of gene expression, yet many of their targets and modes of action remain unknown. In Schizosaccharomyces pombe, one-third of TFs are solely homology-predicted, with few experimentally validated. We created a comprehensive library of 89 endogenously tagged S. pombe TFs, mapping their protein and chromatin interactions using immunoprecipitation-mass spectrometry and chromatin immunoprecipitation sequencing. Our study identified protein interactors for half the TFs, with over a quarter potentially forming stable complexes. We discovered DNA binding sites for most TFs across 2,027 unique genomic regions, revealing motifs for 38 TFs and uncovering a complex regulatory network of extensive TF cross- and autoregulation. Characterization of the largest TF family revealed conserved DNA sequence preferences but diverse binding patterns, and identified a repressive heterodimer, Ntu1/Ntu2, linked to perinuclear gene localization. Our TFexplorer webtool makes all data interactively accessible, offering new insights into TF interactions and regulatory mechanisms with broad biological relevance. HIGHLIGHTSO_LIComprehensive strain library of endogenously tagged S. pombe TFs C_LIO_LIExperimentally determined atlas of TF interactions with proteins and chromatin C_LIO_LITFexplorer web application for interactive exploration of TF interactomes C_LIO_LIIdentification of repressive Nattou complex linked to perinuclear gene localization C_LI

molecular biology↗

bioRxiv2025

Unfolded to Folded: Unraveling the Secrets of Protein Folding with ProteusFold

Protein folding has long been regarded as the "holy grail" of biology, typically demanding large models and massive GPU clusters. This study introduces Pro-teusFold, a compact and interpretable model with only 993,408 parameters that achieves state-of-the-art accuracy on modest hardware with an inference time of 0.0011 seconds. By framing folding as an unfolded-to-folded sequence transformation using a novel structural tokenization, ProteusFold reduces regression complexity while preserving bond connectivity through the concept of "Synapses." It achieves near-atomic fidelity (RMSD 0.24,[A], GDT-TS 99.85) and excels in protein-protein docking with a mean DockQ of 0.7675, with 95.5% of complexes above the 0.23 threshold. Compared to AlphaFold2s Predicted Aligned Error (6.28), ProteusFold attains 0.396, representing an order-of-magnitude gain in positional accuracy. Beyond accuracy and efficiency, the model provides residue-level attribution analyses that highlight biologically significant residues, serving as a preliminary guide for experiments. Furthermore, ProteusFold is the first to provide atomic-level attribution of key electronic and thermal properties, offering deeper insight into folding mechanisms and pinpointing the specific atoms responsible for distinct scenarios. Moreover, a meta-analysis suggests the presence of folding hotspots, where critical residues cluster, revealing new avenues for discovery. Thus, ProteusFold delivers accuracy, interpretability, and efficiency, broadening access to protein-folding research.

molecular biology↗

bioRxiv2019

Mass-spectrometry based proteomics reveals mitochondrial supercomplexome plasticity

Mitochondrial respiratory complex subunits assemble in supercomplexes. Studies of supercomplexes have typically relied upon antibody-based protein quantification, often limited to the analysis of a single subunit per respiratory complex. To provide a deeper insight into mitochondrial and supercomplex plasticity, we combined Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and mass spectrometry to determine the supercomplexome of skeletal muscle from sedentary and exercise-trained mice. We quantified 422 mitochondrial proteins within ten supercomplex bands, in which we showed the debated presence of complex II and V. Upon exercise-induced mitochondrial biogenesis, non-stoichiometric changes in subunits and incorporation into supercomplexes was apparent. We uncovered the dynamics of supercomplex-related assembly proteins and mtDNA-encoded subunits within supercomplexes, as well as the complexes of ubiquinone biosynthesis enzymes and Lactb, a mitochondrial-localized protein implicated in obesity. Our approach can be applied to broad biological systems. In this instance, comprehensively analyzing respiratory supercomplexes illuminates previously undetectable complexity in mitochondrial plasticity. HighlightsO_LIComprehensive quantification of respiratory subunits within supercomplexes C_LIO_LIComplex II and V assemble within supercomplexes C_LIO_LIMitochondrial-encoded subunits display elevated upregulation upon exercise training C_LIO_LIExercise increases ubiquinone biosynthesis enzyme complexes C_LI

molecular biology↗