Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Radiation protection in a cohort of healthcare workers: knowledge, attitude, practices, feelings and IR-exposure in French hospitals.

The number of healthcare workers occupationally exposed to ionizing radiation (IR) is increasing every year. As health effects from exposure to low doses IR have been reported, radiation protection (RP) in the context of occupational activities is a major concern. This study aims to assess the compliance of healthcare workers with RP policies, according to their registered cumulative dose, profession, and perception of radiation self-exposure and associated risk. Every healthcare worker from one of the participating hospitals in France with at least one dosimetric record for each year 2009, 2014, and 2019 in the SISERI registry was included and invited to complete an online questionnaire including information on the worker's occupational exposure, perception of IR-exposure risk and RP general knowledge. Hp(10) doses were provided by the SISERI system. Multivariate logistic regressions were used. Dosimeter wearing and RP practices compliance were strongly associated with 'feeling of being IR-exposed' (OR = 3.69, CI95% 2.04-6.66; OR = 4.60, CI95% 2.28-9.30, respectively). However, none of these factors was associated with RP training courses attendance. The main reason given for non-compliance is unsuitability or insufficient numbers of RP devices. This study provided useful information for RP policies. Making exposed workers aware of their own IR-exposure seems to be a key element to address in RP training courses. This type of questionnaire should be introduced into larger epidemiological studies. Dosimeter wearing and RP practices compliance are associated to feeling being IR-exposed. RP training courses should reinforce workers' awareness of their exposure to IR.

CORDAX web server: An online platform for the prediction and 3D visualization of aggregation motifs in protein sequences.

MOTIVATION: Proteins, the molecular workhorses of biological systems, execute a multitude of critical functions dictated by their precise three-dimensional structures. In a complex and dynamic cellular environment, proteins can undergo misfolding, leading to the formation of aggregates that take up various forms, including amorphous and ordered aggregation in the shape of amyloid fibrils. This phenomenon is closely linked to a spectrum of widespread debilitating pathologies, such as Alzheimer's disease, Parkinson's disease, type-II diabetes, and several other proteinopathies, but also hampers the engineering of soluble agents, as in the case of antibody development. As such, the accurate prediction of aggregation propensity within protein sequences has become pivotal due to profound implications in understanding disease mechanisms, as well as in improving biotechnological and therapeutic applications. RESULTS: We previously developed Cordax, a structure-based predictor that utilizes logistic regression to detect aggregation motifs in protein sequences based on their structural complementarity to the amyloid cross-beta architecture. Here, we present a dedicated web server interface for Cordax. This online platform combines several features including detailed scoring of sequence aggregation propensity, as well as 3D visualization with several customization options for topology models of the structural cores formed by predicted aggregation motifs. In addition, information is provided on experimentally determined aggregation-prone regions that exhibit sequence similarity to predicted motifs, scores, and links to other predictor outputs, as well as simultaneous predictions of relevant sequence propensities, such as solubility, hydrophobicity, and secondary structure propensity. AVAILABILITY: The Cordax webserver is freely accessible at https://cordax.switchlab.org/.

Integrating physics in deep learning algorithms: a force field as a PyTorch module.

MOTIVATION: Deep learning algorithms applied to structural biology often struggle to converge to meaningful solutions when limited data is available, since they are required to learn complex physical rules from examples. State-of-the-art force-fields, however, cannot interface with deep learning algorithms due to their implementation. RESULTS: We present MadraX, a forcefield implemented as a differentiable PyTorch module, able to interact with deep learning algorithms in an end-to-end fashion. AVAILABILITY AND IMPLEMENTATION: MadraX documentation, together with tutorials and installation guide, is available at madrax.readthedocs.io.

Local structural preferences in shaping tau amyloid polymorphism.

Tauopathies encompass a group of neurodegenerative disorders characterised by diverse tau amyloid fibril structures. The persistence of polymorphism across tauopathies suggests that distinct pathological conditions dictate the adopted polymorph for each disease. However, the extent to which intrinsic structural tendencies of tau amyloid cores contribute to fibril polymorphism remains uncertain. Using a combination of experimental approaches, we here identify a new amyloidogenic motif, PAM4 (Polymorphic Amyloid Motif of Repeat 4), as a significant contributor to tau polymorphism. Calculation of per-residue contributions to the stability of the fibril cores of different pathologic tau structures suggests that PAM4 plays a central role in preserving structural integrity across amyloid polymorphs. Consistent with this, cryo-EM structural analysis of fibrils formed from a synthetic PAM4 peptide shows that the sequence adopts alternative structures that closely correspond to distinct disease-associated tau strains. Furthermore, in-cell experiments revealed that PAM4 deletion hampers the cellular seeding efficiency of tau aggregates extracted from Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy patients, underscoring PAM4's pivotal role in these tauopathies. Together, our results highlight the importance of the intrinsic structural propensity of amyloid core segments to determine the structure of tau in cells, and in propagating amyloid structures in disease.

N-glycosylation as a eukaryotic protective mechanism against protein aggregation.

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While posttranslational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biophysical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our findings indicate that, among its many molecular functions, N-glycosylation directly prevents protein aggregation in higher eukaryotes.

LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) ß-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

TDP-43 pathology is associated with increased tau burdens and seeding.

BACKGROUND: Most Alzheimer's Disease (AD) cases also exhibit limbic predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC), besides amyloid-ß plaques and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau (p-tau). LATE-NC is characterized by cytoplasmic aggregates positive for pathological TDP-43 and is associated with more severe clinical outcomes in AD, compared to AD cases lacking TDP-43 pathology TDP-43: AD(LATE-NC-). Accumulating evidence suggests that TDP-43 and p-tau interact and exhibit pathological synergy during AD pathogenesis. However, it is not yet fully understood how the presence of TDP-43 affects p-tau aggregation in symptomatic AD. METHODS: In this study, we investigated the impact of TDP-43 proteinopathy on p-tau pathology with different approaches: histologically, in a human post-mortem cohort (n = 98), as well as functionally using a tau biosensor cell line and TDP-43A315T transgenic mice. RESULTS: We found that AD cases with comorbid LATE-NC, AD(LATE-NC+), have increased burdens of pretangles and/or NFTs as well as increased brain levels of p-tau199, compared to AD(LATE-NC-) cases and controls. The burden of TDP-43 pathology was also correlated with the Braak NFT stages. A tau biosensor cell line treated with sarkosyl-insoluble, brain-derived homogenates from AD(LATE-NC+) cases displayed exacerbated p-tau seeding, compared to control and AD(LATE-NC-)-treated cells. Consistently, TDP-43A315T mice injected with AD(LATE-NC+)-derived extracts also exhibited a more severe hippocampal seeding, compared to the remaining experimental groups, albeit no TDP-43 aggregation was observed. CONCLUSIONS: Our findings extend the current knowledge by supporting a functional synergy between TDP-43 and p-tau. We further demonstrate that TDP-43 pathology worsens p-tau aggregation in an indirect manner and increases its seeding potential, probably by increasing p-tau levels. This may ultimately contribute to tau-driven neurotoxicity and cell death. Because most AD cases present with comorbid LATE-NC, this study has an impact on the understanding of TDP-43 and tau pathogenesis in AD and LATE, which account for the majority of dementia cases worldwide. Moreover, it highlights the need for the development of a biomarker that detects TDP-43 during life, in order to properly stratify AD and LATE patients.

Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding.

There is an arms race between beta-lactam antibiotics development and co-evolving beta-lactamases, which provide resistance by breaking down beta-lactam rings. We have observed that certain beta-lactamases tend to aggregate, which persists throughout their evolution under the selective pressure of antibiotics on their active sites. Interestingly, we find that existing beta-lactamase active site inhibitors can act as molecular chaperones, promoting the proper folding of these resistance factors. Therefore, we have created Pept-Ins, synthetic peptides designed to exploit the structural weaknesses of beta-lactamases by causing them to misfold into intracellular inclusion bodies. This approach restores sensitivity to a wide range of beta-lactam antibiotics in resistant clinical isolates, including those with Extended Spectrum variants that pose significant challenges in medical practice. Our findings suggest that targeted aggregation of resistance factors could offer a strategy for identifying molecules that aid in addressing the global antibiotic resistance crisis.

Mechanisms and pathology of protein misfolding and aggregation.

Despite advances in machine learning-based protein structure prediction, we are still far from fully understanding how proteins fold into their native conformation. The conventional notion that polypeptides fold spontaneously to their biologically active states has gradually been replaced by our understanding that cellular protein folding often requires context-dependent guidance from molecular chaperones in order to avoid misfolding. Misfolded proteins can aggregate into larger structures, such as amyloid fibrils, which perpetuate the misfolding process, creating a self-reinforcing cascade. A surge in amyloid fibril structures has deepened our comprehension of how a single polypeptide sequence can exhibit multiple amyloid conformations, known as polymorphism. The assembly of these polymorphs is not a random process but is influenced by the specific conditions and tissues in which they originate. This observation suggests that, similar to the folding of native proteins, the kinetics of pathological amyloid assembly are modulated by interactions specific to cells and tissues. Here, we review the current understanding of how intrinsic protein conformational propensities are modulated by physiological and pathological interactions in the cell to shape protein misfolding and aggregation pathology.

SNPeffect 5.0: large-scale structural phenotyping of protein coding variants extracted from next-generation sequencing data using AlphaFold models.

BACKGROUND: Next-generation sequencing technologies yield large numbers of genetic alterations, of which a subset are missense variants that alter an amino acid in the protein product. These variants can have a potentially destabilizing effect leading to an increased risk of misfolding and aggregation. Multiple software tools exist to predict the effect of single-nucleotide variants on proteins, however, a pipeline integrating these tools while starting from an NGS data output list of variants is lacking. RESULTS: The previous version SNPeffect 4.0 (De Baets in Nucleic Acids Res 40(D1):D935-D939, 2011) provided an online database containing pre-calculated variant effects and low-throughput custom variant analysis. Here, we built an automated and parallelized pipeline that analyzes the impact of missense variants on the aggregation propensity and structural stability of proteins starting from the Variant Call Format as input. The pipeline incorporates the AlphaFold Protein Structure Database to achieve high coverage for structural stability analyses using the FoldX force field. The effect on aggregation-propensity is analyzed using the established predictors TANGO and WALTZ. The pipeline focuses solely on the human proteome and can be used to analyze proteome stability/damage in a given sample based on sequencing results. CONCLUSION: We provide a bioinformatics pipeline that allows structural phenotyping from sequencing data using established stability and aggregation predictors including FoldX, TANGO, and WALTZ; and structural proteome coverage provided by the AlphaFold database. The pipeline and installation guide are freely available for academic users on https://github.com/vibbits/snpeffect and requires a computer cluster.

Amyloid-like p53 as prognostic biomarker in serous ovarian cancer-a study of the OVCAD consortium.

TP53 is the most commonly mutated gene in cancer and has been shown to form amyloid-like aggregates, similar to key proteins in neurodegenerative diseases. Nonetheless, the clinical implications of p53 aggregation remain unclear. Here, we investigated the presence and clinical relevance of p53 aggregates in serous ovarian cancer (OC). Using the p53-Seprion-ELISA, p53 aggregates were detected in 46 out of 81 patients, with a detection rate of 84.3% in patients with missense mutations. High p53 aggregation was associated with prolonged progression-free survival. We found associations of overall survival with p53 aggregates, but they did not reach statistical significance. Interestingly, p53 aggregation was significantly associated with elevated levels of p53 autoantibodies and increased apoptosis, suggesting that high levels of p53 aggregates may trigger an immune response and/or exert a cytotoxic effect. To conclude, for the first time, we demonstrated that p53 aggregates are an independent prognostic marker in serous OC. P53-targeted therapies based on the amount of these aggregates may improve the patient's prognosis.

Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation.

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.

Enhanced therapeutic window for antimicrobial Pept-ins by investigating their structure-activity relationship.

The overconsumption and inappropriate use of antibiotics is escalating antibiotic resistance development, which is now one of the 10 top threats to global health. Introducing antibiotics with a novel mode of action into clinical use is urgently needed to address this issue. Deliberately inducing aggregation of target proteins and disrupting protein homeostasis in bacteria via amyloidogenic peptides, also called Pept-ins (from peptide interferors), can be lethal to bacteria and shows considerable promise as a novel antibiotic strategy. However, the translation of Pept-ins into the clinic requires further investigation into their mechanism of action and improvement of their therapeutic window. Therefore, we performed systematic structure modifications of 2 previously discovered Pept-ins, resulting in 179 derivatives, and investigated the corresponding impact on antimicrobial potency, cellular accumulation, and ability to induce protein aggregation in bacteria, in vitro aggregation property, and toxicity on mammalian cells. Our results show that both Pept-in accumulation and aggregation of target proteins in bacteria are requisite for Pept-in mediated antimicrobial activity. Improvement of these two parameters can be achieved via increasing the number of arginine residues, increasing Pept-in aggregation propensity, optimizing the aggregate core structure, adopting ß-turn linkers, or forming a disulphide bond. Correspondingly, improvement of these two parameters can enhance Pept-in antimicrobial efficacy against wildtype E. coli BL21 used in the laboratory as well as clinically isolated multidrug-resistant strain E. coli ATCC, A. baumannii, and K. pneumoniae.

Occupational exposure to ionizing radiation in medical staff: trends during the 2009-2019 period in a multicentric study.

OBJECTIVES: Health workers exposed to ionizing radiation account for + 50% of workers exposed to man-made radiation in France. Over the last decade, the use of radiation in medicine has increased due to the introduction of new practices. The EXposition des Professionnels de santE aux RayonnemenTs ioniSants study aims to evaluate and characterize the trends in radiation exposure of health workers in France between 2009 and 2019. METHODS: This retrospective study includes all health workers with at least one dosimetric record in the system for occupational dosimetry registration (Système d'information de la surveillance de l'exposition aux rayonnements ionisants) database for each of the years 2009, 2014, and 2019, in the hospitals included in the study. Individual external doses and socio-professional data were collected. Statistical analyses include descriptions, graphs, and logistic regressions. RESULTS: A total of 1457 workers were included (mean age: 39.8 years, 59% women). The average exposure significantly decreased between 2009 and 2019 (-0.008 mSv/year, p < 0.05). There were large discrepancies in trends according to professions, departments, hospitals, and gender. Over the 10-year study period, radiologic technologists and physicians were the most exposed (0.15 mSv (95%CI 0.14-0.16) and 0.13 mSv (0.06-0.21), respectively), but their exposure tended to decrease. Workers in nuclear medicine departments had the highest radiation exposure (0.36 mSv (0.33-0.39)), which remained stable over time. Thirty-eight percent of recorded doses were nonzero in 2009, decreasing to 20% in 2019. CONCLUSIONS: This study allowed to identify physicians and radiologic technologists in nuclear medicine departments as the most exposed medical workers in France, and to show an overall decrease trend in radiation exposure. This should be instructive for radiation monitoring and safety of exposed medical workers. KEY POINTS: • Radiation exposure of healthcare workers in most medical departments has steadily decreased between 2009 and 2019 in several French hospitals. • The number of zero doses consistently increased during the study period. • Workers in nuclear medicine departments are the most exposed, especially radiologic technologists and physicians.

Reduced Levels of Misfolded and Aggregated Mutant p53 by Proteostatic Activation.

In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.

EndophilinA-dependent coupling between activity-induced calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation.

Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.

Exploiting the intrinsic misfolding propensity of the KRAS oncoprotein.

Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

A guide to studying protein aggregation.

Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un- or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyse protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods.