Biology subjects

Eisen, T. J.

Publications and source records attributed to Eisen, T. J..

8 recordsLinked to original sources

bioRxiv2026

LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation

T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single molecule response is the initiation of calcium influx by activation of PLC{gamma}1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLC{gamma}1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLC{gamma}1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP2 or PIP3 lipids via the PLC{gamma}1 PH domains. We further observe PLC{gamma}1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also revealed a crosstalk mechanism in which the TEC family kinases responsible for PLC{gamma}1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLC{gamma}1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLC{gamma}1 activation through a bimodal switch affecting LAT phosphorylation. Significance StatementT cells are sensitive sensors capable of detecting and responding to trace amounts of foreign antigen. Understanding how they achieve such sensitivity while maintaining accurate antigen discrimination remains a key challenge. Here, through detailed kinetic measurements of PLC{gamma}1 activation, we identify a cross reactivity in which kinases responsible for PLC{gamma}1 phosphorylation also phosphorylate LAT. This creates a bimodal switch controlling LAT phosphorylation levels, which gates PLC{gamma}1 activation from single TCR signals. We suggest this mechanism plays a key role in the signal amplification and noise suppression required for T cells to detect single antigen molecules.

biophysics↗

bioRxiv2025

T cell costimulatory and inhibitory signals differentially modulate LAT condensate nucleation propensity after TCR ligation

Linker for activation of T cells (LAT) is a membrane-surface scaffold protein that assembles key signaling molecules upon T cell receptor (TCR) activation. Recent observations have revealed LAT assembly occurs in discretized condensates traceable to single molecular TCR activation events. Moreover, the condensation process occurs abruptly, but after a distinct delay, indicative of a type of phase transition. Here we examine the effects of costimulatory (CD80, CD86) and inhibitory (PD-L1) ligands on the nucleation propensity of LAT condensation. We utilize single molecule mobility tracking to resolve the moment a pMHC molecule binds to TCR combined with intracellular imaging to measure the time delay between pMHC:TCR binding and nucleation of the corresponding LAT condensate. These delay time distributions reflect the propensity function for stochastic nucleation of the LAT condensate and provide a measure of the effective signal strength from a single, activated TCR. The results reveal that CD28 stimulation by CD80 or CD86 causes a distinct reduction in the mean delay time to LAT condensation while PD-L1 is mildly antagonistic to the process. Collectively, these observations strengthen evidence that probability and timing of the LAT nucleation process itself is tightly associated with signal propagation downstream from individual TCR activation events.

biophysics↗

bioRxiv2025

BTK Autoinhibition Analyzed by High-Throughput SH2 Domain Swaps

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, positioning the SH3 domain in an inhibitory conformation; in Abl, interaction between the kinase domain and a myristoyl group on the N- terminal segment provides the same latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an SH2-kinase latch. To assess autoinhibition in BTK, we generated hundreds of chimeric BTK molecules and measured their fitness using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins demonstrated that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. These results reveal the importance of distributed interactions between the SH2 and kinase domains of BTK in stabilizing the inhibitory conformation, and suggest that the specialized latching mechanisms in Src and Abl kinases may be later evolutionary refinements.

biochemistry↗

bioRxiv2023

Differential Requirement for Dimerization of the Membrane-Binding PH-TH Module of BTK in B cells and T cells

Brutons tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin-homology and tec-homology (PH-TH) domains of BTK are required for signaling. In vitro, dimerization of the PH-TH module strongly stimulates BTK kinase activity. Whether BTK dimerizes in cells via the PH-TH module, and whether this dimerization is necessary for signaling, is unknown. To address this question, we developed high-throughput mutagenesis assays for BTK function in B cells and T cells. We measured the fitness costs for thousands of point mutations in the PH-TH module and kinase domain, allowing us to assess whether dimerization of the PH-TH module and BTK kinase activity are necessary for function. In Ramos B cells we find that neither PH-TH dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations in the PH-TH module that increase membrane adsorption, even at the cost of reduced PH-TH dimerization. In contrast, in Jurkat T cells, we find that BTK signaling depends on both PH-TH dimerization and kinase activity. Evolutionary analysis shows that BTK proteins in fish and lower organisms, like all Tec kinases other than BTK, lack PH-TH dimerization but have active kinase domains. Thus, PH-TH dimerization is not intrinsically required for Tec-kinase activity, and is a special feature that evolved to exert stricter regulatory control on BTK kinase activity as adaptive immune systems gained increased complexity.

biochemistry↗

bioRxiv2022

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance StatementSARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

microbiology↗

bioRxiv2019

MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs

MicroRNAs (miRNAs) specify recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels--even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs.\n\nHighlightsO_LImiRNAs cause accelerated decay of short-tailed target molecules\nC_LIO_LIThis accelerated decay largely prevents accumulation of short-tailed target mRNAs\nC_LIO_LImiRNAs are similarly effective on short-lived and long-lived target mRNAs\nC_LIO_LIIn 3T3 cells, miRNA effects on translation are negligible--even for nascent mRNA\nC_LI

genomics↗

bioRxiv2019

The Dynamics of Cytoplasmic mRNA Metabolism

For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rate constants, which correspond to cytoplasmic lifetimes. Indeed, with few exceptions, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from either endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rate constants of short-tailed mRNAs vary broadly (1000-fold) and are more rapid for short-tailed mRNAs that had previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rate constants to impart a similarly large range in stabilities.\n\nHighlightsO_LImRNAs enter the cytoplasm with diverse intra- and intergenic lengths\nC_LIO_LImRNA deadenylation rates span a 1000-fold range and correspond to mRNA half-lives\nC_LIO_LIAfter their tails become short, mRNAs decay at rates that span a 1000-fold range\nC_LIO_LIMore rapidly deadenylated mRNAs decay more rapidly upon reaching short tail lengths\nC_LI

genomics↗

bioRxiv2018

Transient Hsp90 suppression promotes a heritable change in protein translation

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

cell biology↗