Biology subjects

Martin, A.

Publications and source records attributed to Martin, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Phage growth rate is significantly affected by host-temperature and phage-temperature interactions

Virus growth depends on host performance in any given environment and has a profound impact spanning fields from climate change to human health. We investigated the intrinsic growth rate of two natural G4-like bacteriophages on four clones of their host (E.coli strain C) that differed in their thermal performance, with significant differences in their maximum growth rate and optimum temperature. The intrinsic population growth rate of the G4-like phage isolates was measured on the four host clones at three temperatures, reflective of a low, optimum and high temperature for one of the phage. The phages intrinsic growth rate was significantly affected by host-temperature and phage-temperature interactions, but not by the interaction between host and phage. Across temperatures, phage growth rate was consistently highest on the host clone with the highest maximum growth rate, but was not, contrary to our expectations, lowest on the host with the lowest growth rate. At any given temperature, phage growth rate followed a directional trend with host growth rate, but this trend was not as apparent when looking between temperatures. Our work demonstrates a significant impact of temperature and the host on phage growth rate, but much work remains to be done to disentangle how variation in host performance impacts virus performance across temperatures.

microbiology↗

bioRxiv2025

Stress tolerance of multiple Salmonella enterica strains associated with foodborne outbreaks

Salmonella enterica is a foodborne pathogen commonly found in food processing environments. While control methods such as heat treatment and sanitizers are often used, S. enterica has evolved strategies for survival and persistence to overcome pathogen control. This study assessed 43 outbreak-associated (OA) and non-outbreak associated (NOA) S. enterica isolates from serovars Enteritidis, Heidelberg, Newport, Typhimurium, and monophasic Typhimurium (I 4,[5],12:i:-) for enhanced stress tolerance. Heat shock at 56{degrees} C, minimum inhibitory concentrations (MICs) for sanitizers sodium hypochlorite (NaOCl) and peracetic acid (PAA), and crystal violet microtiter assays were used to evaluate heat tolerance, sanitizer tolerance, and attachment capacity, respectively. Most isolates (n =34/43) carried at least one antimicrobial resistance gene, and nearly half (n =21/43) displayed genotypic and/or phenotypic resistance to ampicillin, ciprofloxacin, or ceftriaxone. Most isolates carried genes conferring resistance to gold (n =43/43) and arsenic (n= 41/43), and tolerance to mercury, copper, and silver was common among monophasic Typhimurium and Heidelberg isolates. Efflux pump qacEdelta1 was detected among eight Heidelberg isolates. We found enhanced stress tolerance (i.e. an unusually high ability to survive and adapt to various environmental stresses) to sanitizers and enhanced attachment capacity, indicating biofilm formation. Isolates evaluated for heat tolerance survived at least 15 min at 56{degrees} C and three survived >60 min. Overall, we found evidence of enhanced tolerance to individual stresses across both OA and NOA S. enterica. There were no strong patterns based upon serovar or OA/NOA status; however, we did find that specific enhanced stress tolerance profiles may have contributed to outbreak characteristics.

microbiology↗

bioRxiv2025

Establishment, maintenance and consequences of inter-individual transcriptional variability for a gene involved in nitrate nutrition in plants

Differences in phenotypes and gene expression are observed between genetically identical plants grown in the same environment. While we now have a good knowledge of the source and consequences of transcriptional differences observed between cells, in particular in unicellular organisms, it is still very scarce when it comes to variability between multicellular organisms. Using NRT2.1, a high affinity nitrate transporter gene, as a model for high inter-individual transcriptional variability, we showed that differences in expression between plants for that gene are established in young seedlings, maintained over time but not transmitted to the next generation. Our results also indicate that these differences in expression could have phenotypic consequences. Indeed, they can explain phenotypic variability for the root growth as well as the amount of nitrate imported by the NRT2.1 protein in the plant. Finally, we found an enrichment for genes involved in photosynthesis among the ones with an expression correlated with NRT2.1 in single seedlings. All in all, our study suggests that a global coordination of the genes involved in the carbon/nitrate balance in plants is established in young seedlings, with differences in this state between plants, and then maintained over time.

plant biology↗

bioRxiv2025

Minos-mediated transgenesis in the pantry moth Plodia interpunctella

Transposon-mediated transgenesis has been widely used to study gene function in Lepidoptera, with piggyBac being the most commonly employed system. However, because the piggyBac transposase originates from a lepidopteran genome, it raises concerns about endogenous activation, remobilization, and silencing of transgenes, thus questioning its suitability as an optimal tool in Lepidoptera. As an alternative, we evaluated the dipteran-derived Minos transposase for stable germline transformation in the pantry moth, Plodia interpunctella. We injected syncytial embryos with transposase mRNA, along with donor plasmids encoding 3xP3::EGFP and 3xP3::mCherry markers of eye and glial tissues. Across multiple experiments, we found that G0 injectees could transmit Minos transgenes through the germline even in the absence of visible marker expression in the soma, and that large mating pools of G0 founders consistently produced transgenic offspring at efficiencies exceeding 10%. Using these methods, we generated transgenic lines with a dual expression plasmid, using 3xP3::mCherry for driving red fluorescence in eyes and glial tissues, as well as the Fibroin-L promoter expressing the recently developed mBaoJin fluorescent protein in the silk glands. This demonstrated the feasibility of screening two pairs of promoter activity in tissues of interest. Collectively, these results--along with previous findings in the silkworm Bombyx mori--demonstrate that Minos achieves robust germline integration of transgenes in Lepidoptera, offering a valuable pathway to the genetic modification of species where the remobilization or suppression of piggyBac elements might be rampant.

genetics↗

bioRxiv2025

Neural activation down to the spinal cord during action language? A transcranial magnetic stimulation and peripheral nerve stimulation study.

Language comprehension is increasingly recognized as extending beyond the traditional linguistic system to engage motor and perceptual processes. This perspective is supported by numerous studies demonstrating that understanding action-related words often induces behavioral and neurophysiological changes in the motor system. However, it remains unclear whether the influence of action language on the motor system is restricted to cortical regions, or whether it also extends to spinal structures, as observed during motor imagery. To address this, we used transcranial magnetic stimulation and peripheral nerve stimulation to assess corticospinal excitability and cortico-motoneuronal transmission, respectively. Fifteen healthy and right-handed volunteers participated in four conditions: (i) rest, (ii) kinesthetic motor imagery of finger and wrist flexion, (iii) reading action sentences, and (iv) reading non-action sentences. As anticipated, corticospinal excitability increased during both kinesthetic motor imagery and action reading compared to rest. Interestingly, while kinesthetic motor imagery also led to the expected increase in cortico-motoneuronal transmission, no such modulation occurred during action reading. These findings suggest that action reading do not modulate the excitability of high-threshold motoneurons at the spinal level, contrary to motor imagery. Further investigation is needed to test whether action reading activate lower-threshold spinal structures, such as interneurons involved in spinal pre-synaptic inhibition.

neuroscience↗

bioRxiv2025

Differential performance of polygenic prediction across traits and populations depending on genotype discovery approach

Polygenic scores (PGS) are widely used to estimate genetic predisposition to complex traits by aggregating the effects of common variants into a single measure. They hold promise in identifying individuals at increased risk for diseases, allowing earlier screening and interventions. Genotyping arrays, commonly used for PGS computation, are affordable and computationally efficient, while whole-genome sequencing (WGS) offers a more comprehensive view of genetic variation. In this study, we compared PGS derived from arrays and WGS across multiple traits to evaluate differences in predictive performance, portability across populations, and computational efficiency. We computed PGS for 10 traits, representing a range of heritability and polygenicity, in the three largest genetic ancestry groups in All of Us (European, African American, Admixed American), trained on multi-ancestry meta-analyses from the Pan-UK Biobank. Using the clumping and thresholding (C+T) method, we found that WGS-based PGS outperformed array-based PRS for highly polygenic traits but showed differentially reduced accuracy for sparse traits in certain populations. With the LD-informed PRS-CS method, we observed overall improved prediction performance compared to C+T, with WGS outperforming arrays across most non-cancer traits. The results obtained using PRS-CS closely align with those derived from pre-trained models in the PGS Catalog, with prediction achieving better performance using WGS than array genotypes for non-sparse traits. To further investigate factors influencing differential prediction performance between array and WGS, we ran simulations varying the proportions of causal SNPs directly captured by the technologies. These demonstrated that the proportion of causal variants genotyped dramatically affects prediction accuracy. Fine-mapping of empirical data supported this concept but also highlighted the importance of reducing non-informative variants for optimal prediction accuracy. In conclusion, while WGS-based PGS generally offer superior predictive power with PRS-CS, the advantage over arrays is context-dependent, varying by trait, population, and the PGS method. The ability to capture causal variants through these technologies largely drives the prediction accuracy. This study provides insights into the complexities and potential advantages of using different genotype discovery approaches for polygenic predictions across populations and informs on strategies to enhance accuracy.

genetics↗

bioRxiv2025

A Bio-inspired Latent TGF-β Conjugated Scaffold Improves Neocartilage Development

In cartilage tissue engineering, active TGF-{beta} is conventionally supplemented in culture medium at highly supraphysiologic doses to accelerate neocartilage development. While this approach enhances cartilage extracellular matrix (ECM) biosynthesis, it further promotes tissue features detrimental to hyaline cartilage function, including the induction of tissue swelling, hyperplasia, hypertrophy, and ECM heterogeneities. In contrast, during native cartilage development, chondrocytes are surrounded by TGF-{beta} configured in a latent complex (LTGF-{beta}), which undergoes cell-mediated activation, giving rise to moderated, physiologic dosing regimens that enhance ECM biosynthesis while avoiding detrimental features associated with TGF-{beta} excesses. Here, we explore a bio-inspired strategy, consisting of LTGF-{beta}-conjugated scaffolds, providing TGF-{beta} exposure regimens that are moderated and uniformly administered throughout the construct. Specifically, we evaluate the performance of LTGF-{beta} scaffolds to improve neocartilage development with bovine chondrocyte-seeded agarose constructs compared to outcomes from active TGF-{beta} media supplementation (MS) at a physiologic 0.3 ng/mL dose (MS-0.3), supraphysiologic 10 ng/mL dose (MS-10), or TGF-{beta} free. For small-size constructs ({emptyset}3x2 mm), LTGF-{beta} scaffolds yield neocartilage that achieves native-matched mechanical properties (800-925 kPa) and sGAG content (6.6%-7.1%), while providing a cell morphology and collagen distribution more reminiscent of hyaline cartilage. LTGF-{beta} scaffolds further afford an optimal chondrogenic phenotype, marked by a 12-to 28-fold reduction of COL-I expression relative to TGF-{beta}-free and a 7-to 17-fold reduction of COL-X expression relative to MS-10. Further, for large-size constructs, which approach the dimensions needed for clinical cartilage repair, LTGF-{beta} scaffolds significantly reduce mechanical and biochemical heterogeneities relative to MS-0.3 and MS-10. Overall, the use of LTGF-{beta} scaffolds improves the composition, structure, material properties, and cell phenotype of neocartilage.

bioengineering↗

bioRxiv2025

Viral-mediated fluorescent labeling of hyaluronan reveals extracellular matrix dynamics in the mouse brain in vivo

The extracellular matrix (ECM) of the brain is primarily composed of the glycan polymer hyaluronan (HA), a core scaffold that nucleates proteoglycans forming a self-assembled matrix that acts as structural framework and signaling hub. Since most of the neural matrix is composed of sugars, development of genetically encoded tags has been limited. Therefore, although several staining protocols exist for ECM in fixed tissue, there are no reliable matrix labels for live imaging. Here we report a viral-mediated fluorescent probe that binds to HA and labels the mouse brain ECM. The vector encodes the HA binding domain from neurocan fused to GFP and an externalization tag (AAV-Ncan-GFP), enabling transduced cells to secrete the fluorescent hyalectan into the extracellular space, thereby labeling HA. We demonstrate stable probe expression in organotypic brain slices, as well as in vivo in the mouse cortex, where it labels both perineuronal nets and interstitial matrix. We validate HA labeling through colocalization with HABP and sensitivity to hyaluronidase, and confirm the probes extracellular localization by shadow imaging. As a proof of concept, we combine AAV-Ncan-GFP with dendritic spine imaging ex vivo and calcium transient imaging in vivo, providing a real-time map of local ECM alongside neural function. The probe enables time-lapse imaging of ECM dynamics in live mice, facilitating longitudinal studies across a wide range of timescales, from minutes to days. The results establish AAV-Ncan-GFP as a valuable tool for real-time observation of brain ECM and a promising resource to explore ECM dynamics and brain function in vivo.

neuroscience↗

bioRxiv2024

Mapping histological and functional maturation of human endocrine pancreas across early postnatal periods

Human endocrine cell differentiation and islet morphogenesis play critical roles in determining islet cell mass and function, but the events and timeline of these processes are incompletely defined. To better understand early human islet cell development and maturation, we collected 115 pediatric pancreata and mapped morphological and spatiotemporal changes from birth through the first ten years of life. Using quantitative analyses and a combination of complementary tissue imaging approaches, including confocal microscopy and whole-slide imaging, we developed an integrated model for endocrine cell formation and islet architecture, including endocrine cell type heterogeneity and abundance, endocrine cell proliferation, and islet vascularization and innervation. We also assessed insulin and glucagon secretory profiles in isolated islet preparations from pediatric donors aged 2 months to 10 years and found a temporal difference in the maturation of insulin secretion compared to glucagon secretion. This comprehensive summary of postnatal and pediatric pancreatic islet development provides a framework for future studies and integration of emerging genetic and genomic data related to islet biology and diabetes risk.

developmental biology↗

bioRxiv2024

Repeated head-exposures to a 5G-3.5 GHz signal do not alter behavior but modify intracerebral gene expression in adult male mice.

The 5th generation (5G) of mobile communications promotes human exposures to electromagnetic fields exploiting the 3.5 GHz frequency band. We have analyzed behaviors, cognitive functions and gene expression in mice submitted to asymmetrical head-exposures to a 5G-modulated 3.5 GHz signal. The exposures were applied 1h daily, 5 days per week over a six-week period, at a specific absorption rate (SAR) averaging 0.19 W/kg over the brain. Locomotor activity in an open-field, object-place and object recognition memories were assessed repeatedly after four weeks of exposure and did not reveal any significant effect on the locomotion/exploration, anxiety level or memory processes. mRNA profiling was performed at the end of the exposure period in two symmetrical areas of the right and left cerebral cortex in which the SAR values were 0.43 and 0.14 W/kg, respectively. We found significant changes in the expression of less than 1% of the expressed genes with over-representations of genes related to glutamatergic synapses. The right cortical area differed from the left one by an over-representation of responsive genes encoded by the mitochondrial genome. Our data show that repeated head-exposures to a 5G-3.5 GHz signal can trigger mild transcriptome alterations without change in memory capacities or emotional state.

neuroscience↗

bioRxiv2024

Genetic basis of an adaptive polymorphism controlling butterfly silver iridescence

Identifying the genes and mutations that drive phenotypic variation and which are subject to selection is crucial for understanding evolutionary processes. Mormon Fritillary butterflies (Speyeria mormonia) exhibit a striking wing color polymorphism throughout their range: typical morphs bear silver spots on their ventral surfaces, and can co-occur with unsilvered morphs displaying a dull coloration1. Through genome-wide association studies in two polymorphic populations, we fine-map this difference in silvering to the 3 region of the transcription factor gene optix. The expression of optix is confined to the unsilvered regions that surround the spots, and these patterns are transformed to a silver identity upon optix RNAi knockdown, implicating optix as a repressor of silver scales in this butterfly. We show that the unsilvered optix haplotype shows signatures of recent selective sweeps, and that this allele is shared with the monomorphic, unsilvered species Speyeria hydaspe, suggesting that introgressions facilitate the exchange of variants of adaptive potential across species. Remarkably, these findings parallel the role of introgressions and cis-regulatory modulation of optix in shaping the aposematic red patterns of Heliconius butterflies2-7, a lineage that separated from Speyeria 45 million years ago8. The genetic basis of adaptive variation can thus be more predictable than often presumed, even for traits that appear divergent across large evolutionary distances. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/628425v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@5d2a9borg.highwire.dtl.DTLVardef@982c74org.highwire.dtl.DTLVardef@8e90b3org.highwire.dtl.DTLVardef@1bddf3c_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

evolutionary biology↗

bioRxiv2024

Atypical scene-selectivity in the retrosplenial complex in individuals with autism spectrum disorder

A small behavioral literature on individuals with autism spectrum disorder (ASD) has shown that they can be impaired when navigating using map-based strategies (i.e., memory-guided navigation), but not during visually guided navigation. Meanwhile, there is neuroimaging evidence in typically developing (TD) individuals demonstrating that the retrosplenial complex (RSC) is part of a memory-guided navigation system, while the occipital place area (OPA) is part of a visually-guided navigation system. A key identifying feature of the RSC and OPA is that they respond significantly more to pictures of places compared to faces or objects - i.e., they demonstrate scene-selectivity. Therefore, we predicted that scene-selectivity would be weaker in the RSC of individuals with ASD compared to a TD control group, while the OPA would not show such a difference between the groups. We used functional MRI to scan groups of ASD individuals and matched TD individuals while they viewed pictures of places and faces and performed a one-back task. As predicted, scene-selectivity was significantly lower in the RSC, but not OPA, in the ASD group compared to the TD group. These results suggest that impaired memory-guided navigation in individuals with ASD may, in part, be due to atypical functioning in the RSC. Lay summaryThe retrosplenial complex (RSC), a cortical region that is part of a neural system that supports our ability to form map-like mental representations of the environment and use them to navigate (i.e., memory-guided navigation), exhibits atypical responses to images of places in individuals with autism spectrum disorder (ASD). These results are a first step towards understanding the neural mechanisms responsible for understudied behavioral impairments in memory-guided navigation in individuals with ASD.

neuroscience↗

bioRxiv2024

Recombinant Expression of Photo-crosslinkable 26S Proteasome Base Subcomplex

The 26S proteasome complex is the hub for regulated protein degradation in the cell. It is composed of two biochemically distinct complexes: the 20S core particle with proteolytic active sites in an internal chamber and the 19S regulatory particle, consisting of a lid and base subcomplex. The base contains ubiquitin receptors and an AAA+ (ATPases associated with various cellular activities) motor that unfolds substrates prior to degradation. The S. cerevisiae base subcomplex can be expressed recombinantly in E. coli and reconstituted into functional 26S proteasomes in vitro, which allows the introduction of unnatural amino acids with novel functions or other mutations that may not be permissible in vivo. Here, we present a method for the introduction of the photo-induced crosslinking amino acid p-benzoyl-L-phenylalanine into the proteasomal base subcomplex. This approach has exciting implications for the study of protein-protein interactions of this complex that mediates the degradation of an incredibly diverse protein pool.

biochemistry↗

bioRxiv2024

Targeted KRAS(G12V) degradation elicits efficient and durable lung adenocarcinoma regression in vivo

Recent drug discovery breakthroughs led to the approval of KRASG12C inhibitors in lung adenocarcinoma (LUAD). Unfortunately, clinical responses remain limited due to rapid resistance onset. Proteolysis-targeting chimeras (PROTACs) have emerged as promising alternatives to traditional inhibition. However, there is limited mechanistic understanding of KRAS degradation in vivo. Here, we developed a preclinical LUAD mouse model and demonstrated that targeted oncogenic KRAS degradation induces rapid tumor regression. Transcriptional, histological, and immunophenotypic analyses revealed a substantial remodeling of the tumor microenvironment. Notably, disease relapse observed during long-term degrader treatment stems from proteolysis machinery dysregulation, indicating resistance mechanisms distinct from those reported upon KRAS inhibition. Our findings highlight the therapeutic potential of KRAS degradation in LUAD, offering insights into cell-intrinsic and extrinsic mechanisms driving durable antitumor responses and supporting further clinical exploration. SIGNIFICANCEGiven the short duration of the clinical responses to KRAS inhibitors, complementary therapies are a dire medical need. Our preclinical findings endorse KRAS degradation as a therapeutic alternative in LUAD, where cell-intrinsic and extrinsic mechanisms drive tumor regression and durable therapeutic responses.

cancer biology↗

bioRxiv2024

Cell-type specific autophagy in human leukocytes

Autophagy is a naturally conserved mechanism crucial for degrading and recycling damaged organelles and proteins to support cell survival. This process slows biological ageing and age-related disease in preclinical models. However, there has been little translation of autophagy to the clinic, and we have identified a lack of measurement tools for physiological human autophagy as a barrier. To address this, we have previously developed a direct measurement tool for autophagy in pooled human peripheral blood mononuclear cells (PBMCs) in the context of whole blood. In order to better understand how autophagy behaves and changes in humans, we measured human autophagic flux using flow cytometry in 19 cell sub-populations in whole blood to retain physiological flux. Autophagic flux was different between different cell types, being highest in B lymphocytes and lowest in T lymphocytes and monocytes. Autophagic flux also varied with sex, being higher in monocytes in females compared with males. In keeping with previous observations in humans, autophagy also increased with ageing at sub-population levels. Importantly, we found that only monocytes - specifically, non-classical monocytes - displayed increased autophagic flux following amino acid withdrawal, underscoring the importance of population selection for measurement of autophagic flux during nutrient restriction studies in humans. Collectively, these data show PBMC population level analysis improves sensitivity of human autophagic flux measurement.

cell biology↗

bioRxiv2024

PITHD1: An Endogenous Inhibitor of the 26S Proteasome During Cellular Dormancy

Cellular dormancy represents a state of regulated growth arrest essential for diverse biological processes, from reproduction to cancer progression. While mechanisms controlling protein synthesis in dormant cells have been identified, how cells regulate protein degradation during dormancy remains unclear. Using zebrafish oocytes, eggs and embryos as a model system, we discovered PITHD1 as an endogenous inhibitor of the 26S proteasome. Our high-resolution cryoEM structure reveals that PITHD1 simultaneously blocks three crucial functional sites on the 19S regulatory particle which are required for ubiquitin recognition, processing, and substrate translocation. This triple-lock mechanism effectively prevents protein degradation in dormant cells. Given PITHD1s evolutionary conservation across species, this mechanism likely represents a general strategy for reversible proteasome regulation during cellular dormancy. Our findings establish a new paradigm for controlling proteostasis in quiescent states.

biochemistry↗

bioRxiv2024

Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1

The 26S proteasome targets many cellular proteins for degradation during general homeostasis, protein quality control, and the regulation of vital processes. A broad range of proteasome-interacting cofactors thereby modulates these functions and aids in substrate degradation. Here, we solved several high-resolution structures of the redox active cofactor TXNL1 bound to the human 26S proteasome at saturating and sub-stoichiometric concentrations by time resolved cryo-EM. We identified distinct binding modes of TXNL1 that depend on the proteasome conformational and ATPase motor states. Together with biophysical and biochemical experiments, our structural studies reveal that the resting-state proteasome prior to substrate engagement with the ATPase motor binds TXNL1 with low affinity and in variable positions on top of the Rpn11 deubiquitinase. In contrast, the actively degrading proteasome shows additional interactions leading to high-affinity TXNL1 binding, whereby TXNL1s C-terminal tail covers the catalytic groove of the Rpn11 deubiquitinase and coordinates the active-site Zn2+. Furthermore, these cryo-EM structures of the degrading proteasome capture the ATPase hexamer in all registers of spiral-staircase arrangements and thus visualize the complete ATP-hydrolysis cycle of the AAA+ motor, indicating temporally asymmetric hydrolysis and conformational changes in bursts during mechanical substrate unfolding and translocation. Remarkably, we catch the proteasome in the act of unfolding the beta-barrel mEos3.2 substrate while the ATPase hexamer is in a particular spiral staircase register. Our findings challenge current models for protein translocation through hexameric AAA+ motors and reveal how the proteasome uses its distinct but broad range of conformational states to coordinate cofactor binding and substrate processing. HighlightsO_LIHigh resolution structures of the redox active cofactor TXNL1 in complex with the human 26S proteasome solved by time-resolved cryo-EM. C_LIO_LITXNL1 binds the catalytic groove of the main proteasomal deubiquitinase Rpn11 and coordinates its active-site Zinc specifically in substrate-degrading states of the proteasome. C_LIO_LIVisualizing a partially unfolded intermediate of the mEos model substrate during processing. C_LIO_LIStructures of the actively degrading human proteasome reveal all spiral-staircase registers of the AAA+ ATPase hexamer with unexpected nucleotide occupancies that indicate asymmetric ATP hydrolysis mechanisms, conformational changes with burst phases, and thus new models for hand-over-hand substrate translocation. C_LI

biochemistry↗

bioRxiv2024

The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome

The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. In addition to targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by directly modulating the conformational switching of the proteasome, yet the underlying mechanisms are unknown. Here, we used biochemical, mutational, and single-molecule FRET-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin-receptor subunit Rpn10, thereby stabilizing the engagement-competent state of the proteasome and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to four-fold faster turnover by the proteasome.

biochemistry↗