Biology subjects

Baldwin, A.

Publications and source records attributed to Baldwin, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Isoform-Specific Localization Diversifies Human MSI2 Function

Musashi-2 (MSI2) is an RNA-binding protein implicated in stem cell regulation and cancer, yet reports of its regulatory activity span translational activation, repression, and effects on mRNA stability, sometimes within the same cellular context. The basis for this diversity has remained unclear. Here, we identify alternative splicing as a determinant of MSI2 regulatory activity, acting through isoform-specific subcellular localization. Using a tethered reporter assay, we show that the canonical MSI2-328 isoform promotes translation without changing mRNA abundance. Truncation and mutation analysis identify a 40-amino-acid region (residues 194-234) as necessary and sufficient for cytoplasmic localization and necessary for translational activation. In contrast, the alternatively spliced MSI2-324 isoform localizes predominantly to the nucleus and fails to promote translation. We map this difference to an 18-amino-acid sequence introduced by an alternative 3 splice acceptor in exon 12; this sequence is sufficient to direct nuclear localization, and its removal restores MSI2-328-like cytoplasmic localization and translational activation. Consistent with these compartmental differences, the two isoforms associate with distinct protein networks, MSI2-328 with translation factors and ribosomal proteins, and MSI2-324 with chromatin and pre-mRNA processing factors. These isoform-specific activities are conserved across cell types, and the relative abundance of MSI2 isoforms shifts toward MSI2-324 in several cancers. Altogether, alternative splicing controls MSI2 subcellular localization, interaction networks, and regulatory output, providing a mechanistic framework for the context-dependent roles of MSI2 in gene regulation.

molecular biology↗

bioRxiv2025

Effects of nonpharmacological manipulations and repeated xanomeline treatment on methamphetamine-vs-food choice in Sprague Dawley and Long Evans rats

The absence of Food and Drug Administration (FDA)-approved pharmacotherapies for methamphetamine use disorder (MUD) highlights the need for preclinical research to understand both the basic biological mechanisms of methamphetamine reinforcement and evaluate novel MUD pharmacotherapies. Recent studies demonstrated that repeated treatment with the muscarinic M1/M4 receptor agonist xanomeline attenuated cocaine self-administration. Whether these xanomeline treatment effects extend to methamphetamine self-administration remains unknown. The first aim established the economic substitutability between methamphetamine and liquid food (i.e., Ensure(R)) using a methamphetamine-vs-food choice procedure in male and female Sprague Dawley (SD) and Long Evans (LE) rats. A within-session methamphetamine choice dose-effect function (0.032-0.32 mg/kg/infusion) was determined daily, and food reinforcer magnitude was manipulated weekly by changing the concentration (0, 10, 32, and 100%) of vanilla-flavored Ensure. Additionally, methamphetamine response requirement (i.e., fixed ratio (FR) 1, 5, 25, 125) was manipulated each week while holding the food FR constant. The second aim determined the effectiveness of repeated 5-day xanomeline (3.2-10 mg/kg, SC) to attenuate methamphetamine choice. Both increasing Ensure concentrations and methamphetamine FR values resulted in rightward shifts in the methamphetamine choice dose-effect function in both SD and LE rats. Repeated 5-day xanomeline treatment significantly decreased methamphetamine choice across all doses tested in LE, but not SD, rats. Time course of xanomeline treatment effectiveness revealed effects were greatest during the first 30 min of choice session. These results demonstrate that methamphetamine and food function as economic substitutes and that xanomeline may warrant further consideration as a MUD pharmacotherapy.

pharmacology and toxicology↗

bioRxiv2025

eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by novel small molecules

Exposure to hypoxia is linked to increased cellular plasticity and enhanced metastasis; effects which are primarily attributed to the transcriptional activation of large gene programs downstream of hypoxia inducible factors (HIFs). However, translational effects in hypoxia, that likely precede transcriptional effects, have remained largely unexplored. Using ribosome-profiling, we uncovered a selective translational response in acute hypoxia that is eIF3d/eIF3e-dependent and controls downstream hypoxic responses including HIF1a accumulation and cellular invasion. We further demonstrated that eIF3e copy number and an eIF3e-expression signature are associated with worsened outcomes for breast cancer patients. Finally, we identified a class of novel small molecules that target eIF3e specifically, reducing the translational response to hypoxia and to ER stress, another stressor that is dependent on eIF3d/eIF3e-mediated translation. Our data uncover critical functions for eIF3d/eIF3e in the hypoxic response and identify a potential means to inhibit stress-induced translation, and potentially plasticity and metastasis, mediated by eIF3e.

cancer biology↗

bioRxiv2024

Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

molecular biology↗

bioRxiv2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of [~]80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing. SIGNIFICANCE STATEMENTOur results highlight the importance of understanding how virally-derived particles function to eliminate unnecessary viral proteins and create more efficacious and easier-to-produce delivery vehicles for therapeutic genome editing.

bioengineering↗

bioRxiv2024

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate as their parental fibroblasts, and the TRAIL produced by iNSCs were naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays showed that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts around 3000-fold greater than treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a new easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

cancer biology↗

bioRxiv2024

Ribosome Profiling Reveals Translational Reprogramming via mTOR Activation in Omacetaxine Resistant Multiple Myeloma

Protein homeostasis is critical to the survival of multiple myeloma (MM) cells. While this is targeted with proteasome inhibitors, mRNA translation inhibition has not entered trials. Recent work illustrates broad sensitivity MM cells to translation inhibitor omacetaxine. We hypothesized that understanding how MM cells become omacetaxine resistant will lead to the development of drug combinations to prevent or delay relapse. We generated omacetaxine resistance in H929 and MM1S MM cell lines and compared them to their parental lines. Resistant lines displayed decreased sensitivity to omacetaxine, with EC50 > 100 nM, compared to parental line sensitivity of 24-54 nM. To adapt to omacetaxine, H929 and MM1S exhibited an increased percentage of multi-nucleated polyaneuploid cells that led to distinct molecular mechanisms of resistance. Interestingly, both resistant lines showed a defect in oncologic potential via extended survival in a MM xenograft model. Since omacetaxine inhibits protein synthesis, we performed both RNA-sequencing and ribosome profiling (Ribo-seq) to identify shared and unique regulatory strategies of resistance. Transcripts encoding translation factors and containing Terminal OligoPyrimidine (TOP) motifs in their 5 UTR were translationally upregulated in both resistant cell lines. The mTOR pathway promotes the translation of TOP motif containing mRNAs. Indeed, mTOR inhibition restored partial sensitivity to omacetaxine in both resistant cell lines. Primary MM cells from patient samples were sensitive to combinations of omacetaxine and mTOR inhibitors rapamycin and Torin 1. These results provide a rational approach for omacetaxine-based combination in patients with multiple myeloma, which have historically shown better responses to multi-agent regimens.

cancer biology↗

bioRxiv2023

In situ spatial reconstruction of distinct normal and pathological cell populations within the human adrenal gland

The human adrenal gland consists of concentrically organized functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from two individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized two APMs in a 72-year-old female. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

genomics↗

bioRxiv2022

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

RNA binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule inhibitor identified as a competitive inhibitor of Musashi(MSI)-RNA interactions. Here we show Ro potently inhibits adrenocortical steroidogenesis and viability independent of MSI2 in multiple cell lines. We identified Ro-interacting proteins using an unbiased proteome-wide approach and discovered it is broadly targeting RBPs. To confirm this finding, we leveraged the large-scale ENCODE data and found a subset of RBPs whose depletion phenocopies Ro inhibition. We conclude that Ro is a promiscuous inhibitor of multiple RBPs, many containing RRM1 domains. Moreover, we provide a general framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

molecular biology↗

bioRxiv2022

In vivo imaging and regression analysis reveal cell-non-autonomous functions of Shroom3 during neural tube closure.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. These cells are physically interconnected via N-cadherin, and mutation of critical genes within relatively small numbers of cells can result in neural tube closure defects due to non-cell autonomous cell behavior defects. Despite this finding, we have a poor understanding of how neuroepithelial cells interact during apical constriction. In our previous paper, we introduced an imaging and analysis paradigm for tracking and quantifying apical constriction during neural tube closure, while also using CRISPR/Cas9 to generate mosaic loss of function of the apical constriction gene shroom3. Here we analyze the behaviors of cells along the mosaic interface of our shroom3 crispant clones, and find that Shroom3 non-cell autonomously regulates apical constriction and N-cadherin localization. Control cells along the interface constrict less, while shroom3 crispant cells along the interface constrict more. Finally, we construct a partial least squares regression (PLSR) model to estimate how both autonomous and non-cell autonomous dynamics of actin and N-cadherin affect apical surface area in both control and shroom3 crispant cells. Overall, our results demonstrate a previously unidentified non-cell autonomous role for Shroom3 in neural tube closure.

developmental biology↗

bioRxiv2021

Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis

Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer end on-binding to sialoside sugars was revealed contrasting with extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

biophysics↗

bioRxiv2021

αB-crystallin affects the morphology of Aβ(1-40) aggregates

B-crystallin (ABC) is a human small heat shock protein that is strongly linked to Alzheimers disease (AD). In vitro, it can inhibit the aggregation and amyloid formation of a range of proteins including A{beta}(1-40), a primary component of AD amyloid plaques. Despite the strong links, the mechanism by which ABC inhibits amyloid formation has remained elusive, in part due to the notorious irreproducibility of aggregation assays involving preparations of A{beta}-peptides of native sequence. Here, we present a recombinant expression protocol to produce native A{beta}(1-40), devoid of any modifications or exogenous residues, with yields up to 4 mg/L E. coli. This material provides highly reproducible aggregation kinetics and, by varying the solution conditions, we obtain either highly ordered amyloid fibrils or more disordered aggregates. Addition of ABC slows the aggregation of A{beta}(1-40), and interferes specifically with the formation of ordered amyloid fibrils, favouring instead the more disordered aggregates. Solution-state NMR spectroscopy reveals that the interaction of ABC with A{beta}(1-40) depends on the specific aggregate morphology. These results provide mechanistic insight into how ABC inhibits the formation of amyloid fibrils. HighlightsO_LIProtocol for production of native recombinant A{beta}(1-40) C_LIO_LIAmyloid formation under physiological conditions is highly reproducible C_LIO_LIBoth ordered fibrils and disordered aggregates can be reliably formed C_LIO_LIB-crystallin specifically inhibits amyloid fibril assembling, favouring disordered aggregates C_LI eTOC blurbMuller et al. introduce a protocol for the highly reproducible production of amyloid from native A{beta}(1-40) and determine that the human chaperone ABC specifically destabilises them in favour of disordered aggregates. NMR shows that ABC can distinguish between aggregate morphologies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/433908v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@fe31fborg.highwire.dtl.DTLVardef@8367aforg.highwire.dtl.DTLVardef@10c42a3org.highwire.dtl.DTLVardef@1eaddeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Angiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in our knowledge related to precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate siRNA screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

genomics↗

bioRxiv2021

Global analysis of cell behavior and protein localization dynamics reveals region-specific functions for Shroom3 and N-cadherin during neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure This approach elucidates new differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3 and demonstrates the ability of tissue-level imaging and analysis to generate cell-biological mechanistic insights into neural tube closure.

developmental biology↗

bioRxiv2020

A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

RNA-sequencing is a powerful and increasingly prevalent method to answer biological questions. Depletion of ribosomal RNA (rRNA), which accounts for 80% of total RNA, is an extremely important step to increase the power of RNA-seq. Selection for polyadenylated RNA is a commonly used approach that excludes rRNA, as well as, important non-polyadenylated RNAs, such as histones, circular RNAs, and many long noncoding RNAs. Commercial methods to deplete rRNA are cost-prohibitive and the gold standard method is no longer available as a standalone kit. Alternative non-commercial methods suffer from inconsistent depletion. Through careful characterization of all reaction parameters, we developed an optimized RNaseH-based depletion of human rRNA. Our method exhibited comparable or better rRNA depletion compared to commercial kits at a fraction of the cost and across a wide-range of input RNA amounts.

molecular biology↗

bioRxiv2019

Dysregulation of HSP27 oligomerization and interactions by a neuropathy-causing mutation in the C-terminal IPV motif

HSP27 (HSPB1) is a systemically expressed human small heat-shock protein that forms large, dynamic oligomers and functions in various aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of the disease is triggered by the P182L mutation within the highly conserved IxI/V motif of HSP27. Here, we observed that the P182L variant of HSP27 lacks the ability to prevent the aggregation of client proteins and formed significantly larger oligomers both in vitro and in vivo. NMR spectroscopy revealed that the P182L IxI/V motif binds its -crystallin domain with significantly lower association rate, and thus affinity, rendering the binding site more available for other interactors. We identified 22 IxI/V-containing proteins that are known to interact with HSP27 and could therefore bind with enhanced affinity to the P182L variant. We validated this hypothesis through co-immunoprecipitation experiments, revealing that the IxI/V motif-bearing co-chaperone BAG3 indeed binds with higher affinity to the P182L variant. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27, and highlight the general importance of the IxI/V motif and its role in protein-protein interaction networks.

biochemistry↗

bioRxiv2019

Local frustration determines loop opening in protein-protein association

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein binding sites. Here we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point towards an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

biophysics↗

bioRxiv2019

REMI-seq: Development of methods and resources for functional genomics in Dictyostelium

Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in cell, developmental and evolutionary biology. However, its utility is hampered by the inefficiency with which sequence, transcriptome or proteome variation can be linked to phenotype. To address this, we have developed methods (REMI-seq) to (1) generate a near genome-wide resource of individual mutants (2) allow large-scale parallel phenotyping. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Therefore, these methods and resources provide a step change for high throughput gene discovery in a key model system, and the study of genes affecting traits associated with higher eukaryotes.

genomics↗