Biology subjects

James, L. C.

Publications and source records attributed to James, L. C..

18 recordsLinked to original sources

bioRxiv2025

Whole body MondoA deletion protects against diet-induced obesity through uncontrolled multi-organ substrate utilization and futile cycling

ObjectiveDelineating the nodal control points that maintain whole-body energy homeostasis is critical for understanding potential treatments of obesity and cardiometabolic diseases. The nutrient-sensing transcription factor MondoA is a regulator of skeletal muscle fuel storage, where muscle-specific inhibition improves glucose tolerance and insulin sensitivity. However, the role of MondoA in whole body energy metabolic homeostasis is not understood. MethodsGeneralized MondoA knockout (gKO) mice were generated and assessed for glucose tolerance and insulin sensitivity, body composition, energy expenditure, cold tolerance, and tissue specific transcriptional changes in response to high fat diet. Complementary studies in cultured human adipocytes assessed the impact of MondoA deficiency on substrate utilization and lipolysis. ResultsgKO mice are protected from diet-induced obesity and insulin resistance, through increased whole body energy expenditure. gKO mice exhibit reduced brown and inguinal white adipose tissue mass, without evidence of beiging. The gKO mice are hyperlactatemic and isolated MondoA-deficient adipocytes have increased 2-deoxyglucose uptake and glycolytic function. Lastly, gKO mice and KO adipocytes display increased circulating glycerol relative to free fatty acids in response to adrenergic stimulus consistent with elevated re-esterification. However, this phenotype is not recapitulated in adipocyte-specific KO mice. ConclusionsMondoA deficiency alters cellular sensing of nutrient availability and storage/utilization mechanisms. In the whole-body setting, this results in increased energy expenditure, potentially related to increased glucose uptake and glycolytic flux driving glycerol synthesis to supply high rates of lipolysis and lipid re-esterification. These results suggest that MondoA functions to maintain fuel storage and when lost, inter-organ futile cycling ensues. O_FIG O_LINKSMALLFIG WIDTH=140 HEIGHT=200 SRC="FIGDIR/small/680559v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ccf42dorg.highwire.dtl.DTLVardef@b2da28org.highwire.dtl.DTLVardef@1081aforg.highwire.dtl.DTLVardef@1b2168d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO 1) Global MondoA deficiency drives 2) tissue glucose uptake which in skeletal muscle is 3) converted and excreted as lactate, while in adipose tissue 4) triglyceride re-esterification requires 5) de novo glycerol synthesis to feed into the futile cycle. C_FIG

physiology↗

bioRxiv2025

The host protein cyclophilin A inhibits HIV-1 nuclear entry by decreasing capsid elasticity

Binding of the host protein cyclophilin A (CypA) to the HIV-1 capsid exerts a variety of effects on infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. For several HIV-1 mutants, CypA binding inhibits nuclear entry by an unknown mechanism. We recently demonstrated that HIV-1 cores are elastic and that HIV-1 mutants with inelastic capsids are impaired for nuclear entry and infection of nondividing cells. Here we show that CypA prevents infection of nondividing cells by such mutants and inhibits their entry into the nucleus. CypA binding to mutant cores further reduced their elasticity in vitro, and this effect was reversed by suppressor mutations that restored nuclear entry. We suggest that HIV-1 nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during traversal of the nuclear pore.

microbiology↗

bioRxiv2025

Vaccine-induced antibodies are sufficient to limit Salmonella infection in the absence of complement or macrophages.

Antibodies to Salmonella Typhimurium (STm) can protect against infection. Understanding better how antibodies, complement, and leukocytes interplay can support vaccine development. We used an Outer Membrane Vesicle (OMV) vaccine against STm to study the in vivo function of anti-STm antibodies. Using intravital microscopy, we found that upon challenge, OMV-specific antibodies promote STm uptake by spleen and liver macrophages, with neutrophils rarely capturing the bacteria. Clodronate-liposome depletion of monocytic cells reveals that these cells help prevent antigen dissemination. After vaccination and challenge of C1q, C3, C4, and C5-deficient mice, all mice except C3-deficient were protected by OMV immunization. C3-deficient mice failed to mount significant germinal center and plasma cell responses; however, after the adoptive transfer of immune sera, they had lower bacterial burdens than controls. In vitro, we showed that antibodies enhance bacterial capture in macrophages. Thus, antibodies alone are sufficient to reduce bacterial burdens, but they cooperate with complement and macrophages to maximize their functions in vivo.

immunology↗

bioRxiv2025

The central pore of HIV-1 capsomers promotes sustained stability of the viral capsid

The HIV-1 capsid, which orchestrates several key post-entry events to facilitate infection in target cells, is composed of hexamers and pentamers (capsomers) of the capsid (CA) protein arranged in a closed, conical structure known as the capsid that protects the viral RNA genome and replicative enzymes reverse transcriptase (RT) and integrase (IN). Each capsomer contains a central pore lined with rings of positively charged amino acid side chains - Arg-18 (R18) and Lys-25 (K25). The R18 and K25 rings drive capsid assembly by binding the host polyanion inositol hexakisphosphate (IP6) and are proposed to mediate the import of dNTPs into the capsid to facilitate reverse transcription. Here we demonstrate that the R18 ring can be functionally replaced by the introduction of a mutation (N21K) that establishes a new electropositive ring within the central pore. In contrast with previous studies in which R18 mutants were unable to adapt in culture, the N21K mutation facilitated the acquisition of second-site compensatory mutations that restored near-WT fitness to viral mutants lacking the R18 ring. Comparative analysis of several central pore mutants lacking the R18 ring revealed that particle infectivity was not correlated with IP6 binding or capsid assembly, but rather with capsid stability and key post-entry events including reverse transcription and nuclear entry. Our results indicate that the central pore plays critical roles in both the assembly of capsids and their sustained stability post-entry.

microbiology↗

bioRxiv2024

Antibody-dependent Intracellular neutralisation by TRIM21 is potentiated by HOIP and linear ubiquitin chains

Antibody-dependent intracellular neutralisation (ADIN) promotes the rapid proteasomal degradation of viruses and other large substrates in the cytosol. It is dependent on detection of intracellular virus-bound antibodies by the Fc receptor and E3 ligase TRIM21, followed by disassembly of the virus by the unfoldase VCP/p97. It is not known how VCP is recruited to TRIM21. We performed a limited siRNA knock-down screen of known VCP adaptors to determine their involvement in ADIN. Knock-down of HOIP, the only ubiquitin E3 ligase capable of generating linear ubiquitin chains, resulted in impaired virus neutralisation. HOIPIN-8, a HOIP inhibitor, showed concentration-dependent reduction in virus neutralisation. We found that the activity of HOIP in ADIN is dependent on its ubiquitin binding domains and its PUB domain which recruits VCP. Knock-down of OTULIN, the only deubiquitinating enzyme that exclusively cleaves linear ubiquitin chains, potentiated neutralisation of the virus. Our results expand the role of HOIP and linear ubiquitin chains in proteostasis.

immunology↗

bioRxiv2022

Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6 and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple co-factors to a single site is coordinated, all remain to be elucidated. Here, we have resolved the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to high resolution. We have determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology. Significance statementHIV-1 particles contain a characteristic, conical capsid that shields the genome from the cellular immune system and recruits cellular proteins to direct the capsid to the nucleus. The cone forms from hexamers of CA protein, and twelve pentamers that accommodate curvature. We obtained detailed 3D models of pentamers and hexamers at positions on capsid surfaces with different curvatures. We find two places in CA that switch conformation according to the local capsid curvature and whether CA is in a pentamer or hexamer. We also obtained models of CA bound to peptides from cellular proteins. The data show how switches in CA help it form a cone shape, and interact differently with cellular proteins at different positions on the cone surface.

microbiology↗

bioRxiv2022

Trim-Away degrades lysine-less substrates without requiring ligase autoubiquitination

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while TRIM21 activation results in ubiquitination of both ligase and substrate, autoubiquitination is regulatory and not required for substrate degradation. Substrate binding stimulates N-terminal RING autoubiquitination by the E2 Ube2W, but when inhibited by N-terminal acetylation this prevents neither substrate ubiquitination nor degradation and has no impact on TRIM21 antiviral activity. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Substrate ubiquitination mediates degradation but Trim-Away efficiently degrades lysine-less substrates, suggesting a non-canonical ubiquitination mechanism explains its broad substrate specificity.

molecular biology↗

bioRxiv2022

Macrophage activation of cGAS and TRIM5 distinguish pandemic and non-pandemic HIV

Pandemic viruses remain a global threat to health and economics but how they adapt to become pandemic remains poorly understood. Here we compare pandemic HIV-1(M) and non-pandemic HIV-(O) and HIV-2 strains finding that non-pandemic HIV replicate poorly in myeloid cell models due to activation of cGAS and TRIM5, and ensuing antiviral responses. We use phylogenetics and viral capsid structural biology to define specific differences between pandemic and non-pandemic HIV capsids and demonstrate that their genetic reversal in HIV-1(M) mutants causes TRIM5, cGAS and innate immune activation. We propose a model in which the parental lineage of pandemic HIV-1(M) has uniquely evolved a dynamic capsid that avoids activation of cGAS and TRIM5 to establish cloaked replication in myeloid cells. The unique adaptations of the pandemic virus lineage suggests a role in effective human-to-human transmissibility and highlight the importance of avoiding innate immune activation during pandemic human-to-human viral transmission.

immunology↗

bioRxiv2021

Tau assemblies enter the cytosol of neurons in a cholesterol sensitive manner

Accumulating evidence supports a prion-like mechanism in the spread of assembled tau in neurodegenerative diseases. Prion-like spread is proposed to require the transit of tau assemblies to the interior of neurons in order to seed aggregation of native, cytosolic tau. This process is poorly understood and remains largely hypothetical. Here, we develop sensitive techniques to quantify the cytosolic entry of tau in real-time. We find that tau does not promote its own entry but, rather, is wholly dependent on cellular machinery. We find that entry to the widely used reporter cell line HEK293 requires clathrin whereas entry to neurons does not. Cholesterol depletion or knockdown of cholesterol transport protein Niemann-Pick type C1 in neurons renders cells highly vulnerable to cytosolic entry and seeded aggregation. Our findings establish entry as the rate-limiting step in seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/449238v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1849023org.highwire.dtl.DTLVardef@13129eorg.highwire.dtl.DTLVardef@2b4736org.highwire.dtl.DTLVardef@15038ac_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2021

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

immunology↗

bioRxiv2021

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies. Whilst untreated slices displayed no overt signs of pathology, exposure to tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau only arises at supra-physiological concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

neuroscience↗

bioRxiv2021

SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

immunology↗

bioRxiv2020

Sequences in the cytoplasmic tail of SARS-CoV-2 spike facilitate syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

cell biology↗

bioRxiv2020

SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

neuroscience↗

bioRxiv2020

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

microbiology↗

bioRxiv2020

Substrate-induced clustering activates Trim-Away of pathogens and proteins

Trim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

molecular biology↗

bioRxiv2020

Learning to count: determining the stoichiometry of bio-molecular complexes using fluorescence microscopy and statistical modelling

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which a given biomolecule is differentially labelled with spectrally distinct fluorescent dyes (label A or B), which are then mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology. The statistical models used in our analysis are available here: https://github.com/sophiamersmann/molecular-counting, the raw data used for molecular counting can be found here: 10.5281/zenodo.3955142.

bioinformatics↗

bioRxiv2020

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

biochemistry↗