Biology subjects

Carroll, E. C.

Publications and source records attributed to Carroll, E. C..

9 recordsLinked to original sources

bioRxiv2025

Characterisation of the dual roles of senescent-like T cells that arise during healthy and unhealthy ageing

Ageing is accompanied by progressive remodelling of the immune system, but chronic metabolic disease may accelerate this process and drive qualitatively distinct forms of immune dysfunction. Here, we used type 2 diabetes (T2D) as a model of unhealthy immune ageing to identify a distinct population of CD8 TEMRA cells that accumulates in older individuals with T2D. These cells were highly differentiated, oligoclonally expanded and had shorter telomeres, consistent with an increased replicative history and premature senescence-like state. Unlike conventional TEMRA cells, which can preserve cytotoxic function through acquisition of NK-cell receptors, T2D-associated TEMRA cells showed reduced surface expression of KLRG1, NKG2D and NKG2A together with defective receptor recycling. TGF{beta}1 was elevated in T2D and reproduced several features of this phenotype in vitro, including increased TEMRA differentiation, reduced NK-receptor expression, altered receptor trafficking and induction of p21. Functionally, these cells displayed reduced TCR-triggered degranulation, correlating with impaired cytotoxicity and altered tissue distribution in individuals with T2D. Rather than providing effective immune surveillance, the accumulation of these highly differentiated TEMRA cells with diminished effector capacity may compromise immune function. Together, these findings identify a distinct senescent-like CD8 TEMRA state linking metabolic inflammation to dysregulated T cell differentiation.

immunology↗

bioRxiv2024

Methods for high throughput discovery of fluoroprobes that recognize tau fibril polymorphs

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimers disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery.

biochemistry↗

bioRxiv2023

Interaction with the membrane-anchored protein CHIC2 constrains the ubiquitin ligase activity of CHIP

Maintenance of cellular health requires the proper regulation of E3 ubiquitin ligases. The E3 ligase CHIP is canonically regulated by its interactions with the molecular chaperones Hsp70 and Hsp90, which focus CHIPs ubiquitination activity on misfolded proteins. Here, we report a chaperone-independent interaction of CHIP with the membrane-anchored protein CHIC2, which strongly attenuates CHIPs ligase activity. We show that CHIC2 outcompetes abundant, cytosolic chaperones through its exquisite CHIP selectivity, rather than through enhanced affinity. In proteomic experiments, we find that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIPs functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.

biochemistry↗

bioRxiv2023

Conformationally Adaptive Dyes Enable Protein-Adaptive Differential Scanning Fluorometry

Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding. One-Sentence SummaryNext generation protein-adaptive DSF (paDSF) enables rapid and general measurements of protein stability and dynamics.

biochemistry↗

bioRxiv2021

Two distinct classes of co-chaperones compete for the EEVD motif in heat shock protein 70 (Hsp70) to tune its activity

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions (PPIs) with many co-chaperones. One hotspot for co-chaperone binding is the EEVD motif that is found at the extreme C-terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat (TPR) domain co-chaperones, such as the E3 ubiquitin ligase CHIP, and Class B J-domain proteins (JDPs), such as DnaJB4. Although complexes between Hsp70-CHIP and Hsp70-DnaJB4 are both important for chaperone functions, the molecular determinants that dictate the competition between these co-chaperones are not clear. Using a collection of EEVD-derived peptides, we find that DnaJB4 binds to the IEEVD motif of Hsp70s, but not the related MEEVD motif of cytoplasmic Hsp90s. Then, we explored which residues are critical for binding to CHIP and DnaJB4, revealing that they rely on some shared features of the IEEVD motif, such as the C-terminal carboxylate. However, they also had unique preferences, especially at the isoleucine position. Finally, we observed a functionally important role for competition between CHIP and DnaJB4 in vitro, as DnaJB4 can limit the ubiquitination activity of the Hsp70-CHIP complex, while CHIP suppresses the chaperone activities of Hsp70-DnaJB4. Together, these results suggest that the EEVD motif has evolved to support diverse PPIs, such that competition between co-chaperones could help guide whether Hsp70-bound proteins are folded or degraded.

biochemistry↗

bioRxiv2020

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity--and, by extension, the myriad effects of ubiquitination on substrate proteins--arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrates C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words) Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a proteins energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate-ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

biophysics↗

bioRxiv2019

Ubiquitination modulates a protein energy landscape site-specifically with consequences for proteasomal degradation

Cellular environments modulate protein energy landscapes to drive important biology, where small perturbations are consequential for biological signaling, allostery, and other vital processes. The energetic effects of ubiquitination are interesting due to its potential influence on degradation by the 26S proteasome, which requires intrinsically flexible or unstructured initiation regions that many known proteasome substrates lack. We generated proteins with natively attached, isopeptide-linked ubiquitin in structured domains to assess the energetic changes contributed by ubiquitin and how such changes manifest at the proteasome. Ubiquitination at sensitive sites destabilizes the native structure, and thereby increases the rate of degradation for substrates containing unstructured initiation regions. Importantly, this ubiquitination can even induce those requisite regions in well-folded proteins for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

biochemistry↗

bioRxiv2019

GATA3 controls mitochondrial biogenesis in primary human CD4+ T cells during DNA damage

Introductory paragraphGATA binding protein 3 (GATA3) has traditionally been regarded as a lineage-specific transcription factor that drives the differentiation of CD4+ T helper (Th) 2 cells. However, increasing evidence shows that GATA3 is involved in a myriad of processes such as immune regulation, proliferation and maintenance in other T cell and non-T cell lineages. We show here a previously unknown mechanism utilized by CD4+ T cells to increase mitochondrial mass in response to DNA damage through the binding of GATA3, AMP-activated protein kinase (AMPK), peroxisome-proliferator-activated receptor {gamma} co-activator-1 (PGC1), nuclear factor erythroid 2-related factor 2 (NRF2) and superoxide dismutase 3 (SOD3) to the DNA damage repair (DDR) component ATR. These findings extend the pleotropic nature of GATA3 and highlight the potential for GATA3-targeted cell manipulation for clinical interventions.

immunology↗

bioRxiv2019

Mitochondrial mass governs the extent of T cell senescence

The susceptibility of human CD4+ and CD8+ T cells to senesce differs, with CD8+ T cells acquiring an immunosenescent phenotype faster than their CD4+ T cell compartment. We show here that it is the inherent difference in mitochondrial content that drives this phenotype, with senescent human CD4+ T cells displaying a higher mitochondrial mass. The loss of mitochondria in the senescent human CD8+ T cells has knock-on consequences for nutrient usage, metabolism and function. Mitochondrial dysfunction has been linked to both cellular senescence and ageing, however it is still unclear whether mitochondria play a causal role in senescence. Our data shows that reducing mitochondrial function in human CD4+ T cells, through the addition of low dose rotenone, causes the generation of a CD4+ T cell with a CD8+ -like phenotype. Therefore we wish to propose that it is the inherent metabolic stability that governs the susceptibility to an immunosenescent phenotype.

immunology↗