Teaching protein structure and function through molecular visualization.

The function of proteins is governed by their three-dimensional structure. This structure is determined by the chemical characteristics and atomic interactions of amino acids. Students of biochemistry, with a particular focus on protein chemistry, benefit from looking at protein structures and understanding how proteins are built and fold. Due to their three-dimensional nature, static two-dimensional representations in textbooks can be limiting to student learning. Here, we developed a series of tutorials that introduce students to molecular graphics software. The students are challenged to apply the software to look at proteins and to get a deeper understanding of how amino acid properties are linked to structure. We also familiarize students with some of the latest tools in computational structural biology. Students performed the tutorials with visual enthusiasm and reported general satisfaction in being able to visualize theoretical concepts learned during lectures. We further stimulated student engagement by allowing space for self-exploration. We share the tutorial instructions for other teachers to build on them, and we also offer suggestions for further improvement based on student feedback. In summary, we present a series of tutorials aimed at students of an advanced course in protein biochemistry to enable them to explore the universe of protein structures and how those relate to function.

The scaffolding function of LSD1 controls DNA methylation in mouse ESCs.

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Salmonella infection impacts host proteome thermal stability.

Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

Mechanical Loading Modulates AMPK and mTOR Signaling in Muscle Cells.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Prognostic impact of variant histology in bladder cancer: Would early and aggressive treatment shift the paradigm?

INTRODUCTION: Bladder cancer (BC) is an increasingly frequent malignancy worldwide. Several variant histologies (VH) have been described in BC with a distinct clinical behavior. OBJECTIVES: This study aims to assess the prognostic impact of VH in BC, comparing its outcomes to pure urothelial carcinoma PUC in both non-muscle invasive (NMIBC) and muscle-invasive (MIBC) settings. METHODS: We included patients with primary BC, comparing those with VH with those with PUC, with an age and sex-matched proportion of 1:3, considering stage at diagnosis, recurrence-free, progression-free, and overall survival (OS). A total of 616 patients were included in the study, (460 UC and 151 VH). RESULTS: After first TURBT, MIBC was present in 99 (64.1%) of patients with VH, and 95 (20.6%) with UC (p<0.001). Concerning NMIBC, we observed higher rates of progression to MIBC amid patients with VH (p=0.009). Nodal involvement (p=0.020) and metastatic disease (p<0.001) were significantly higher within the VH group. A higher OS was observed among patients with NMIBC of PUC (p<0.001). There were no statistically significant differences of metastasis-free survival and OS between VH and UC groups within the MIBC setting. CONCLUSION: We confirmed that VH presents a more aggressive clinical course compared to PUC. An earlier radical treatment within the NMIBC setting could increase the oncological outcomes of the VH patients.

Systematic analysis of drug combinations against Gram-positive bacteria.

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface ( https://apps.embl.de/combact/ ).

D-amino acids signal a stress-dependent run-away response in Vibrio cholerae.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release D-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that D-arginine and D-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These D-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either D-arginine or D-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these D-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that D-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Deep thermal profiling for detection of functional proteoform groups.

The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.

Differences in motor unit behavior during isometric contractions in patients with diabetic peripheral neuropathy at various disease severities.

The aim of this study was to determine whether HD-sEMG is sensitive to detecting changes in motor unit behavior amongst healthy adults and type 2 diabetes mellitus (T2DM) patients presenting diabetic peripheral neuropathy (DPN) at different levels. Healthy control subjects (CON, n = 8) and T2DM patients presenting no DPN symptoms (ABS, n = 8), moderate DPN (MOD, n = 18), and severe DPN (SEV, n = 12) performed isometric ankle dorsiflexion at 30 % maximum voluntary contraction while high-density surface EMG (HD-sEMG) was recorded from the tibialis anterior muscle. HD-sEMG signals were decomposed, providing estimates of discharge rate, motor unit conduction velocity (MUCV), and motor unit territory area (MUTA). As a result, the ABS group presented reduced MUCV compared to CON. The groups with diabetes presented significantly larger MUTA compared to the CON group (p < 0.01), and the SEV group presented a significantly lower discharge rate compared to CON and ABS (p < 0.01). In addition, the SEV group presented significantly higher CoVforce compared to CON and MOD. These results support the use of HD-SEMG as a method to detect peripheral and central changes related to DPN.

A Comparison of Delay-and-Add and Maximum Likelihood Estimation for Velocity-Selective Recording Using Multi-Electrode Cuffs.

Extracting information from the peripheral nervous system with implantable devices remains a significant challenge that limits the advancement of closed-loop neural prostheses. Linear electrode arrays can record neural signals with both temporal and spatial selectivity, and velocity selective recording using the delay-and-add algorithm can enable classification based on fibre type. The maximum likelihood estimation method also measures velocity and is frequently used in electromyography but has never been applied to electroneurography. Therefore, this study compares the two algorithms using in-vivo recordings of electrically evoked compound action potentials from the ulnar nerve of a pig. The performance of these algorithms was assessed using the velocity quality factor (Q-factor), computational time and the influence of the number of channels. The results show that the performance of both algorithms is significantly influenced by the number of channels in the recording array, with accuracies ranging from 77% with only two channels to 98% for 11 channels. Both algorithms were comparable in accuracy and Q-factor for all channels, with the delay-and-add having a slight advantage in the Q-factor.

Transient Glycolytic Complexation of Arsenate Enhances Resistance in the Enteropathogen Vibrio cholerae.

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we found that the cholera pathogen V. cholerae exhibits increased resistance to arsenate compared to closely related enteric pathogens. Such resistance is promoted not by ArsC-dependent reduction of arsenate to arsenite but by an operon encoding an arsenate transporter (ArsJ), an alternative glyceraldehyde 3-phosphate dehydrogenase (VarG), and a putative, uncharacterized phosphatase (VarH). Mechanistically, we demonstrate that V. cholerae detoxifies arsenate by complexing it with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). 1As3PG is not transported outside the cell; instead, it is subsequently dissociated by VarH to enable extrusion of free arsenate through ArsJ. Collectively, this study proposes a novel mechanism for arsenate detoxification, entirely independent of arsenate reduction and arsenite extrusion, that enhances V. cholerae resistance to this metalloid compared to other enteric pathogens.

Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

Drug Target Identification in Tissues by Thermal Proteome Profiling.

Drug target deconvolution can accelerate the drug discovery process by identifying a drug's targets (facilitating medicinal chemistry efforts) and off-targets (anticipating toxicity effects or adverse drug reactions). Multiple mass spectrometry-based approaches have been developed for this purpose, but thermal proteome profiling (TPP) remains to date the only one that does not require compound modification and can be used to identify intracellular targets in living cells. TPP is based on the principle that the thermal stability of a protein can be affected by its interactions. Recent developments of this approach have expanded its applications beyond drugs and cell cultures to studying protein-drug interactions and biological phenomena in tissues. These developments open up the possibility of studying drug treatment or mechanisms of disease in a holistic fashion, which can result in the design of better drugs and lead to a better understanding of fundamental biology.

High-throughput functional characterization of protein phosphorylation sites in yeast.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Transcriptional and Post-Transcriptional Polar Effects in Bacterial Gene Deletion Libraries.

Single-gene deletions can affect the expression levels of other genes in the same operon in bacterial genomes. Here, we used proteomics for 133 Escherichia coli gene deletion mutants and transcriptome sequencing (RNA-seq) data from 71 mutants to probe the extent of transcriptional and post-transcriptional effects of gene deletions in operons. Transcriptional effects were common on genes located downstream of the deletion and were consistent across all operon members, with nearly 40% of operons showing greater than 2-fold up- or downregulation. Surprisingly, we observed an additional post-transcriptional effect that leads to the downregulation of the gene located directly downstream of the targeted gene. This effect was correlated with their intergenic distance, despite the ribosome binding site of the gene downstream remaining intact during library construction. Overall, the data presented can guide future library construction and highlight the importance of follow-up experiments for assessing direct effects of single-gene deletions in operons. IMPORTANCE Single-gene deletion libraries have allowed genome-wide characterization of gene function and interactions. While each mutant intends to disrupt the function of a single gene, it can unintentionally target other genes, such as those located in the same operon as the deletion. The extent to which such polar effects occur in deletion libraries has not been assessed. In this work, we use proteomics and transcriptomics data to show that transcript level changes lead to nearly 40% of deletions in operons affecting the protein levels of genes located downstream by at least 2-fold. Furthermore, we observed a post-transcriptional effect on the gene located directly downstream of the deletion. These results can guide the design of future gene deletion libraries and emphasizes the importance of follow-up work when linking genotypes to phenotypes.

The rise of proteome-wide biophysics.

While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of structural and stability proteomic technologies to derive new insights in biology and medicine.