Biology subjects

Kutuzov, M.

Publications and source records attributed to Kutuzov, M..

8 recordsLinked to original sources

bioRxiv2025

Deciphering the dark side of histone ADP-ribosylation: what structural features of damaged nucleosome regulate the activities of PARP1 and PARP2

Poly(ADP-ribose) polymerases are critical enzymes contributing to regulation of numerous cellular processes, including DNA repair. Within the PARP family, PARP1 and PARP2 primarily facilitate PARylation in the nucleus, particularly responding to genotoxic stress. The activity of PARPs is influenced by the nature of DNA damage and multiple protein partners, with HPF1 being the important one. Forming a joint active site with PARP1 (PARP2), HPF1 contributes to histone PARylation and following chromatin remodelling during genotoxic stress events. This study elucidates interrelation between the presence and location of a one-nucleotide gap within the nucleosome core particle (NCP) and PARP activities in automodification and heteromodification of histones. Utilizing a combination of classical biochemical methods with fluorescence-based technique and a single-molecule mass photometry approach, we have shown that the NCP architecture impacts the efficiency and pattern of histone ADP-ribosylation and binding to the histones-associated damaged DNA more significantly for PARP2 than for PARP1. Analysis based on existing studies of HPF1-dependent ADP-ribosylome and NCP structural dynamics allows to suggest that the DNA damage location and the conformational flexibility of histone tails modulated by post-translational modifications are crucial for delineating the distinct roles of PARP1 and PARP2 during genotoxic stress responses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/635606v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2762a9org.highwire.dtl.DTLVardef@1be7cb8org.highwire.dtl.DTLVardef@ee1b14org.highwire.dtl.DTLVardef@33e99b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Using CombiCells, a platform enabling titration and combinatorial display of cell surface ligands, to investigate the sensitivity and costimulatory requirements of TCRs and CARs

Understanding how cellular decisions by receptor/ligand interactions at cell/cell interface has been challenging because it is difficult to independently vary the surface density of multiple ligands. Here, we exploit the SpyCatcher/SpyTag split-protein system for rapid combinatorial display of native ligands on cells (Combicells). We use this platform to assess T cell antigen sensitivity and the impact of T cell co-stimulation/co-inhibition receptors. The TCR displayed much greater sensitivity to pMHC than CARs and BiTES did to CD19. While TCR sensitivity was greatly enhanced by CD2 ligand, CAR sensitivity to CD19 was primarily but more modestly enhanced by LFA-1 ligand. Lastly, we show that the PD-1/ligand engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor/ligand interactions at cell/cell interfaces. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/545075v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@d1ec95org.highwire.dtl.DTLVardef@33c0forg.highwire.dtl.DTLVardef@5bfed7org.highwire.dtl.DTLVardef@1b80ee2_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryUsing CombiCells, a platform for the combinatorial display of cell surface ligands, to compare T cell antigen sensitivity mediated by TCRs, CARs, and BiTEs and its dependence on co-stimulation/co-inhibition receptor ligands

immunology↗

bioRxiv2021

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can re-direct T cells to target abnormal cells but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here we show that, while CARs have a >100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting-cells (APCs), they have nearly identical sensitivity when antigen is presented as purified protein on artificial surfaces. We next measured the impact of engaging accessory receptors (CD2, LFA-1, CD28, CD27, 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved TCR antigen sensitivity by 125 and 22-fold, respectively, but only improved CAR sensitivity by <5-fold. This differential effect of CD2 and LFA-1 engagement on TCR versus CAR sensitivity was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3{varepsilon} subunit (also known as a TRuC), and fully restored by exchanging the CAR variable domains with the TCR{beta} variable domains (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors, and suggest approaches to increase sensitivity.

immunology↗

bioRxiv2021

Missense variants in human ACE2 modify binding to SARS-CoV-2 Spike

SARS-CoV-2 infection begins with the interaction of the SARS-CoV-2 Spike (Spike) and human angiotensin-converting enzyme 2 (ACE2). To explore whether population variants in ACE2 might influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities for Spike receptor binding domain through surface plasmon resonance (SPR). We discovered variants that enhance and reduce binding, including two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro ({Delta}{Delta}G = {+/-} 0.59 0.08 kcal mol-1) is often seen in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg ({Delta}{Delta}G = 0.26 0.09 kcal mol-1) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. Carriers of these alleles may be more susceptible to infection or severe disease and these variants may influence the global epidemiology of Covid-19. We also identified three rare ACE2 variants that strongly inhibited (p.Glu37Lys, {Delta}{Delta}G = -1.33 {+/-} 0.15 kcal mol-1 and p.Gly352Val, predicted {Delta}{Delta}G = -1.17 kcal mol-1) or abolished (p.Asp355Asn) Spike binding. These variants may confer resistance to infection. Finally, we calibrated the mCSM-PPI2 {Delta}{Delta}G prediction algorithm against our SPR data, give new predictions for all possible ACE2 missense variants at the Spike interface and estimate the overall burden of ACE2 variants on Covid-19 phenotypes.

genetics↗

bioRxiv2020

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

T cells use their T cell receptors (TCRs) to discriminate between peptide MHC (pMHC) ligands that bind with different affinities but precisely how different remains controversial. This is partly because the affinities of physiologically relevant interactions are often too weak to measure. Here, we introduce a surface plasmon resonance protocol to measure ultra-low TCR/pMHC affinities (KD ~ 1000 M). Using naive, memory, and blasted human CD8+ T cells we find that their discrimination power is unexpectedly low, in that they require a large >100-fold decrease in affinity to abolish responses. Interestingly, the discrimination power reduces further when antigen is presented in isolation on artificial surfaces but can be partially restored by adding ligands to CD2 or LFA-1. We were able to fit the kinetic proof-reading model to our data, yielding the first estimates for both the time delay (2.8 s) and number of biochemical steps (2.67). The fractional number of steps suggest that one of the proof-reading steps is not easily reversible.

immunology↗

bioRxiv2020

The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context

The repair processes regulation including base excision repair (BER) is implemented by a cellular signal PARylation catalysed by PARP1 and PARP2. Despite intensive studies, it is far from clear how BER is regulated by PARPs and how the roles are distributed between the PARPs. Here, we investigated the effects of PARP1, PARP2 and PARylation on activities of the main BER enzymes (APE1, Pol{beta} and LigIII) in combination with XRCC1 in the nucleosomal context. We constructed nucleosomes with midward- or outward-oriented damage. It was concluded that in most cases, the presence of PARP1 leads to the suppression of the activities of APE1, Pol{beta}, and to a lesser extent LigIII. PARylation by PARP1 attenuated this effect to various degrees. PARP2 had an influence predominantly on the last stage of BER: DNA sealing. Nonetheless, PARylation by PARP2 led to Pol{beta} inhibition and to significant stimulation of LigIII activities in a NAD+-dependent manner.

biochemistry↗

bioRxiv2020

Determination of the molecular reach of the protein tyrosine phosphatase SHP-1

Immune receptor signalling proceeds by the binding (or tethering) of enzymes to their cytoplasmic tails before they catalyse reactions on substrates within reach. This is the case for the enzyme SHP-1 that, upon tethering to the inhibitory receptor PD-1, dephosphorylates membrane substrates to suppress T cell activation. Precisely how tethering regulates SHP-1 activity is incompletely understood. Here, we use surface plasmon resonance to measure binding, catalysis, and molecular reach for PD-1 tethered SHP-1 reactions. We find that the reach of PD-1--SHP-1 complexes is dominated by the 13.0 nm reach of SHP-1 itself. This is longer than an estimate from the structure of the allosterically active conformation (5.3 nm), suggesting that SHP-1 explores multiple active conformations. Using modelling, we show that when uniformly distributed, PD-1--SHP-1 complexes can only reach 15% of substrates but this increases to 90% when they are co-clustered. When within reach, we show that membrane recruitment increases the activity of SHP-1 by a 1000-fold increase in local concentration. The work highlights how molecular reach regulates the activity of membrane-recruited SHP-1 with insights applicable to other membrane-tethered reactions. Significance statementImmune receptors transduce signals by recruiting (or tethering) cytoplasmic enzymes to their tails at the membrane. When tethered, these enzymes catalyse reactions on other substrates to propagate signalling. Precisely how membrane tethering regulates enzyme activity is incompletely understood. Unlike other tethered reactions, where the enzyme tethers to the substrate, the substrate in this case is a different receptor tail. Therefore, the ability of the receptor-tethered enzyme to reach a substrate can be critical in controlling reaction rates. In this work, we determine the molecular reach for the enzyme SHP-1 and the receptor PD-1 to which it can tether, and show how molecular reach controls receptor signalling.

biophysics↗

bioRxiv2020

Human CD8+ T cells exhibit a shared antigen threshold for different effector responses

T cells recognising cognate pMHC antigens become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesised to exhibit different antigen dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the antigen. Here, using systematic experiments in a reductionist system, where primary human CD8+ T cell blasts are stimulated by recombinant pMHC antigen alone, we show that different inflammatory cytokines have comparable antigen dose thresholds across a 25,000-fold variation in affinity. Although co-stimulation by CD28, CD2, and CD27 increased cytokine production in this system, the antigen threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous antigen presenting cells equivalent thresholds were also observed for cytokine production and killing. These findings imply a simple phenotypic model of TCR signalling where multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of antigen recognition, where the chance factor of antigen dose and affinity do not provide any additional response-specific information.

immunology↗