Biology subjects

Williams, D.

Publications and source records attributed to Williams, D..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

The physical form of microbial ligands bypasses the need for dendritic cell migration to stimulate adaptive immunity

A central paradigm of immunology is that the innate immune system first detects infectious agents in peripheral tissues, shortly after a pathogen has breached an epithelial barrier. This detection event is mediated by pattern recognition receptors in phagocytes, which then migrate to draining lymph nodes (dLNs), where information of a microbial encounter is conveyed to T and B lymphocytes to generate adaptive immunity. Through the study of fungal moieties, we present data that challenge this model. We found that soluble fungal polysaccharides are immunosilent in the periphery, but become potent immunogens in the dLN. These ligands completely bypass the need of phagocyte migration and, instead, directly activate an immune response that is most similar to those that typify viral infections. These data establish a class of microbial products that violate a central tenet of the immunological lexicon and illustrate that the physical form (not just the chemical structure) impacts innate and adaptive immunity.

immunology↗

bioRxiv2020

Similar visual perception in GCaMP6 transgenic mice despite differences in learning and motivation

To study mechanisms of perception and cognition, neural measurements must be made during behavior. A goal of the Allen Brain Observatory is to map activity in distinct cortical cell classes during visual processing and behavior. Here we characterize learning and performance of five GCaMP6-expressing transgenic lines trained on a visual change detection task. We used automated training procedures to facilitate comparisons across mice. Training times varied, but most transgenic mice learned the task. Motivation levels also varied across mice. To compare mice in similar motivational states we subdivided sessions into over-, under-, and optimally motivated periods. When motivated, the pattern of perceptual decisions were highly correlated across transgenic lines, although overall d-prime was lower in one line labeling somatostatin inhibitory cells. These results provide important context for using these mice to map neural activity underlying perception and behavior.

neuroscience↗

bioRxiv2020

JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

BackgroundCachexia (CX), a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitor factor (LIF) which induces CX. We characterized how LIF promotes CX-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues. MethodsCX was induced in vivo with C26c20 colon adenocarcinoma cells or recombinant LIF administration in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. CX was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These activated adipocytes were processed for lipolysis, immunoblot analysis, and RT-PCR. ResultsTumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing CX-associated adipose wasting and anorexia. We identified two JAK inhibitors that block cytokine-mediated adipocyte lipolysis and IL-6 induction using an in vitro CX lipolysis assay. JAK inhibitors administered to in vivo colon cancer CX mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the CX serum cyto/adipokine signature, 3) a delay in colon cancer CX-associated anorexia and adipose loss, and 4) an improvement in overall survival. ConclusionsJAK inhibitors suppress cytokine-associated adipose loss and anorexia in multiple in vitro and in vivo models of cancer CX.

cancer biology↗

bioRxiv2020

Innate immune gene expression in Acropora palmata is consistent despite variance in yearly disease events

Coral disease outbreaks are expected to increase in prevalence, frequency and severity due to climate change and other anthropogenic stressors. This is especially worrying for the Caribbean branching Acropora palmata which has already seen an 80% decrease in its coral cover, with this primarily due to disease. Despite the importance of this species, there has yet to be a characterization of its transcriptomic response to disease exposure. In this study we provide the first transcriptomic analysis of 12 A. palmata genotypes, and their symbiont Symbiodiniaceae, exposed to disease in 2016 and 2017. Year was the primary driver of sample variance for A. palmata and the Symbiodiniaceae. Lower expression of ribosomal genes in the coral, and higher expression of transmembrane ion transport genes in the Symbiodiniaceae indicate that the increased virulence in 2017 may have been due to a dysbiosis between the coral and Symbiodiniaceae. We also identified a conserved suite of innate immune genes responding to the disease challenge that was activated in both years. This included genes from the Toll-like receptor and lectin pathways, and antimicrobial peptides. Co-expression analysis identified a module positively correlated to disease exposure rich in innate immune genes, with D-amino acid oxidase, a gene implicated in phagocytosis and microbiome homeostasis, as the hub gene. The role of D-amino acid oxidase in coral immunity has not been characterized but holds potential as an important enzyme for responding to disease. Our results indicate that A. palmata mounts a similar immune response to disease exposure as other coral species previously studied, but with unique features that may be critical to the survival of this keystone Caribbean species.

molecular biology↗

bioRxiv2020

Transfer of hepatocellular microRNA regulates cytochrome P450 2E1 in renal tubular cells

Extracellular microRNAs have been demonstrated to have the ability to enter kidney tubular cells and modify gene expression. We have used a Dicer-hepatocyte-specific microRNA conditional knock-out (Dicer-CKO) mouse to investigate functional microRNA transfer from liver to kidney under physiological conditions and in the context of drug toxicity. Dicer-CKO mice demonstrated a time-dependent decrease in the hepatocyte-derived microRNA, miR-122, in the kidney in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNAs increased expression and activity of the miR-122 target - cytochrome (CYP) P450 2E1 - in the kidney. Serum extracellular vesicles (ECVs) from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin proximal tubular cell toxicity. miR-122 also increased in urinary ECVs during hepatotoxicity in humans. Transfer of microRNA was not restricted to liver injury - we detected miR-499 release with murine cardiac injury, and this correlated with an increase in the kidney. In summary, a physiological transfer of microRNA to the kidney exists, which is increased by liver injury. Regulation of renal drug response due to signalling by microRNA of hepatic origin represents a new paradigm for understanding and preventing nephrotoxicity.

pharmacology and toxicology↗

bioRxiv2019

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogens obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis (DPA) to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.\n\nAuthor SummaryMycobacterium leprae, the obligate human pathogen is uncultivable in axenic growth medium, and this hinders research on this pathogen, and the pathogenesis of leprosy. The development of novel therapeutics relies on the understanding of growth, survival and metabolism of this bacterium in the host, the knowledge of which is currently very limited. Here we reconstructed a metabolic network of M. leprae-GSMN-ML, a powerful in silico tool to study growth and metabolism of the leprosy bacillus. We demonstrate the application of GSMN-ML to identify the metabolic pathways, and metabolite classes that M. leprae utilizes during intracellular growth.

microbiology↗

bioRxiv2019

A Petascale Automated Imaging Pipeline for Mapping Neuronal Circuits with High-throughput Transmission Electron Microscopy

Serial-section electron microscopy is the method of choice for studying cellular structure and network connectivity in the brain. We have built a pipeline of parallel imaging using transmission electron automated microscopes (piTEAM) that scales this technology and enables the acquisition of petascale datasets containing local cortical microcircuits. The distributed platform is composed of multiple transmission electron microscopes that image, in parallel, different sections from the same block of tissue, all under control of a custom acquisition software (pyTEM) that implements 24/7 continuous autonomous imaging. The suitability of this architecture for large scale electron microscopy imaging was demonstrated by acquiring a volume of more than 1 mm3 of mouse neocortex spanning four different visual areas. Over 26,500 ultrathin tissue sections were imaged, yielding a dataset of more than 2 petabytes. Our current burst imaging rate is 500 Mpixel/s (image capture only) per microscope and net imaging rate is 100 Mpixel/s (including stage movement, image capture, quality control, and post processing). This brings the combined burst acquisition rate of the pipeline to 3 Gpixel/s and the net rate to 600 Mpixel/s with six microscopes running acquisition in parallel, which allowed imaging a cubic millimeter of mouse visual cortex at synaptic resolution in less than 6 months.

neuroscience↗

bioRxiv2019

Arfgef1 haploinsufficiency in mice alters neuronal endosome composition and decreases membrane surface postsynaptic GABAA receptors

ARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1fs) in mice. Arfgef1fs/+ pups exhibit signs of developmental delay, and Arfgef1fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABAA receptors (GABAAR) are reduced in Arfgef1fs/+ neurons with an accompanying decrease in GABAAR-containing recycling endosomes in cell body. Arfgef1fs/+ neurons also display differences in the relative ratio of Arf6+:Rab11+:TrfR+ recycling endosomes. Although the GABAAR-containing early endosomes in Arfgef1fs/+ neurons are comparable to wildtype, Arfgef1fs/+ neurons show an increase in GABAAR-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABAAR results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility.\n\nHighlightsO_LIArfgef1fs/+ mice have lower seizure threshold but no spontaneous seizure.\nC_LIO_LIArfgef1fs/+ neurons show reduced dendritic surface GABAAR.\nC_LIO_LIArfgef1fs/+ neurons have decreased GABAAR-containing recycling endosome accompanied with an increase in GABAAR-containing lysosomes.\nC_LI

genetics↗

bioRxiv2019

Determining organ weight toxicity with Bayesian causal models: Improving on the analysis of relative organ weights

Regulatory authorities require animal toxicity tests for new chemical entities. Organ weight changes are accepted as a sensitive indicator of chemically induced organ damage, but can be difficult to interpret because changes in organ weight might reflect chemically-induced changes in overall body weight. A common solution is to calculate the relative organ weight (organ to body weight ratio), but this inadequately controls for the dependence on body weight - a point made by statisticians for decades, but which has not been widely adopted. The recommended solution is an analysis of covariance (ANCOVA), but it is rarely used, possibly because both the method of statistical correction and the interpretation of the output may be unclear to those with minimal statistical training. Using relative organ weights can easily lead to incorrect conclusions, resulting in poor decisions, wasted resources, and an ethically questionable use of animals. We propose to cast the problem into a causal modelling framework as it directly assesses questions of scientific interest, the results are easy to interpret, and the analysis is simple to perform with freely available software. Furthermore, by taking a Bayesian approach we can model unequal variances, control for multiple testing, and directly provide evidence of safety.

pharmacology and toxicology↗

bioRxiv2019

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the P berghei life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatio-temporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is therefore necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of{Delta} kinesin-8X parasites reveals modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression supporting a role in cell division.\n\nAuthor SummaryKinesins are microtubule-based motors that play key roles in intracellular transport, cell division and motility. Members of the Kinesin-8 family contribute to chromosome alignment during cell division in many eukaryotes. However, the roles of kinesins in the atypical cell division in Plasmodium, the causative agent of malaria, is not known. In contrast to many other eukaryotes, Plasmodium proliferates by endomitosis, in which genome replication and division occur within a nucleus bounded by a persistent nuclear envelope. We show that the Plasmodium genome encodes only nine kinesins and we further investigate the role of Kinesin-8X throughout the Plasmodium life cycle using biochemical and gene targeting approaches. We show that Plasmodium Kinesin-8X has microtubule-based motility and depolymerization activity. We also show that Kinesin-8X is probably localized on putative MTOCs and spindles during cell division in most of the stages of P. berghei life cycle. By gene deletion we demonstrate that Kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of{Delta} kinesin-8X parasites reveals modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that Kinesin-8X is a molecular motor that plays essential roles during endomitosis in oocyst development in the mosquito, contributing to parasite transmission.

cell biology↗

bioRxiv2019

Liver-Chip: Reproducing Human and Cross-Species Toxicities

Nonclinical rodent and non-rodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans contributing to drug failures in the clinic. Here we applied microengineered Organ-on-Chip (Organ-Chip) technology to design rat, dog, and human Liver-Chips containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chips detected diverse phenotypes of liver toxicity including hepatocellular injury, steatosis, cholestasis, and fibrosis as well as species-specific toxicities when treated with tool compounds. Multi-species Liver-Chips may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.\n\nOne Sentence SummaryMicroengineered Organ-Chip technology has been used to design rat, dog and human Liver-Chips that recapitulate species-specific liver toxicities.

pharmacology and toxicology↗

bioRxiv2018

Multi-plane Imaging of Neural Activity From the Mammalian Brain Using a Fast-switching Liquid Crystal Spatial Light Modulator

We report a novel two-photon fluorescence microscope based on a fast-switching liquid crystal spatial light modulator and a pair of galvo-resonant scanners for large-scale recording of neural activity from the mammalian brain. The utilized imaging technique is capable of monitoring large populations of neurons spread across different layers of the neocortex in awake and behaving mice. During each imaging session, all visual stimulus driven somatic activity could be recorded in the same behavior state. We observed heterogeneous response to different types of visual stimuli from ~ 3,300 excitatory neurons reaching from layer II/III to V of the striate cortex.

bioengineering↗

bioRxiv2018

Growth substrate may influence biofilm susceptibility to antibiotics

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

microbiology↗

bioRxiv2018

ITPase Deficiency Causes Martsolf Syndrome With a Lethal Infantile Dilated Cardiomyopathy

Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Through genetic analysis of 85 unrelated \"mutation negative\" probands referred with Martsolf syndrome we identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). The probands reported here each presented with a lethal and highly distinctive disorder; Martsolf syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rl/dl) into RNA and DNA. In Itpa-null cells, dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA but this was not associated with detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in lymphoblastoid RNA from an affected individual. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA - and by implication rI production - correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.\n\nAuthor SummaryNucleotide triphosphate bases containing inosine, ITP and dITP, are continually produced within the cell as a consequence of various essential biosynthetic reactions. The enzyme inosine triphosphate pyrophosphatase (ITPase) scavenges ITP and dITP to prevent their incorporation into RNA and DNA. Here we describe two unrelated families with complete loss of ITPase function as a consequence of disruptive mutations affecting both alleles of ITPA, the gene that encodes this protein. Both of the families have a very distinctive and severe combination of clinical problems, most notably a failure of heart muscle that was lethal in infancy or early childhood. They also have features of another rare genetic disorder affecting the brain and the eyes called Martsolf syndrome. We could not detect any evidence of dITP accumulation in genomic DNA from the affected individuals. A low but detectable level of inosine was present in the mitochondrial DNA but this did not have any obvious detrimental effect. The inosine accumulation in RNA was detectable in the patient cells. We made both cellular and animal models that were completely deficient in ITPase. Using these reagents we could show that the highest level of inosine accumulation into RNA was seen in the embryonic mouse heart. In this tissue more than 1 in 400 bases in all RNA in the cell was inosine. In normal tissues inosine is almost undetectable using very sensitive assays. The inosine accumulation did not seem to be having a global effect on the balance of RNA molecules or proteins.

genetics↗

bioRxiv2018

A large-scale, standardized physiological survey reveals higher order coding throughout the mouse visual cortex

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of neural activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes cortical activity from nearly 60,000 neurons collected from 6 visual areas, 4 layers, and 12 transgenic mouse lines from 221 adult mice, in response to a systematic set of visual stimuli. Using this dataset, we reveal functional differences across these dimensions and show that visual cortical responses are sparse but correlated. Surprisingly, responses to different stimuli are largely independent, e.g. whether a neuron responds to natural scenes provides no information about whether it responds to natural movies or to gratings. We show that these phenomena cannot be explained by standard local filter-based models, but are consistent with multi-layer hierarchical computation, as found in deeper layers of standard convolutional neural networks.

neuroscience↗

bioRxiv2018

A mechanistic hydro-epidemiological model of liver fluke risk

The majority of existing models for predicting disease risk in response to climate change are empirical. These models exploit correlations between historical data, rather than explicitly describing relationships between cause and response variables. Therefore, they are unsuitable for capturing impacts beyond historically observed variability and cannot be employed to assess interventions. In this study, we integrate environmental and epidemiological processes into a new mechanistic model, taking the widespread parasitic disease of fasciolosis as an example. The model simulates environmental suitability for disease transmission, explicitly linking the parasite life-cycle to key weather-water-environment conditions. First, using epidemiological data, we show that the model can reproduce observed infection levels in time and space over two case studies in the UK. Second, to overcome data limitations, we propose a calibration approach based on Monte Carlo sampling and expert opinion, which allows constraint of the model in a process-based way, including a quantification of uncertainty. Finally, comparison with information from the literature and a widely-used empirical risk index shows that the simulated disease dynamics agree with what has been traditionally observed, and that the new model gives better insight into the time-space patterns of infection, which will be valuable for decision support.

epidemiology↗

bioRxiv2018

Genetic meta-analysis identifies 10 novel loci and functional pathways for Alzheimer’s disease risk

Late onset Alzheimers disease (AD) is the most common form of dementia with more than 35 million people affected worldwide, and no curative treatment available. AD is highly heritable and recent genome-wide meta-analyses have identified over 20 genomic loci associated with AD, yet only explaining a small proportion of the genetic variance indicating that undiscovered loci exist. Here, we performed the largest genome-wide association study of clinically diagnosed AD and AD-by-proxy (71,880 AD cases, 383,378 controls). AD-by-proxy status is based on parental AD diagnosis, and showed strong genetic correlation with AD (rg=0.81). Genetic meta analysis identified 29 risk loci, of which 9 are novel, and implicating 215 potential causative genes. Independent replication further supports these novel loci in AD. Associated genes are strongly expressed in immune-related tissues and cell types (spleen, liver and microglia). Furthermore, gene-set analyses indicate the genetic contribution of biological mechanisms involved in lipid-related processes and degradation of amyloid precursor proteins. We show strong genetic correlations with multiple health-related outcomes, and Mendelian randomisation results suggest a protective effect of cognitive ability on AD risk. These results are a step forward in identifying more of the genetic factors that contribute to AD risk and add novel insights into the neurobiology of AD to guide new drug development.

genetics↗

bioRxiv2018

Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

Small heat-shock proteins (sHSP) are molecular chaperones that bind and sequester partially and globally unfolded states of their client proteins. Of paramount importance to their physiological roles is the assembly into large oligomers, which for mammalian sHSP are polydisperse and undergo subunit exchange. The flexibility and dynamic nature of these oligomers mediates functional regulation by phosphorylation and underpins the deleterious effects of disease-linked mutations. Previously, we discovered that the archaeal Hsp16.5, which natively forms ordered and symmetric 24-subunit oligomers, can be engineered to transition to an ordered and symmetric 48-subunit oligomer by insertion of a peptide from human HspB1 (Hsp27) at the junction of the N-terminal and -crystallin domains. Here, we carried out a detailed analysis of the determinants of Hsp16.5 oligomeric plasticity by altering the sequence and length of the inserted peptide. Utilizing light scattering, blue native gel electrophoresis, native mass spectrometry and electron microscopy, we uncovered the existence of an array of oligomeric states (30 to 38 subunits) that can be populated as a consequence of different insertions. These oligomers are intermediate states on the assembly pathway of the 48-subunit oligomer as two of the variants can concurrently form 24-subunit or 30-38 subunit polydisperse oligomers. Polydisperse Hsp16.5 oligomers displayed higher affinity to a model client protein consistent with a general mechanism for recognition and binding that involves increased access of the hydrophobic N-terminal region. Our findings, which integrate structural and functional analyses from evolutionarily-distant sHSP, support a model wherein the modular architecture of these proteins encodes motifs of oligomer polydispersity, dissociation and expansion to achieve functional diversity and regulation.

biochemistry↗