PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors ...binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
SIRT6 is a member of the Sirtuin family of histone deacetylases that has been implicated in inflammatory, aging and metabolic pathways. Some of its actions have been suggested to be via physical ...interaction with NFκB and HIF1α and transcriptional regulation through its histone deacetylase activity. Our previous studies have investigated the histone deacetylase activity of SIRT6 and explored its ability to regulate the transcriptional responses to an inflammatory stimulus such as TNFα. In order to develop a greater understanding of SIRT6 function we have sought to identify SIRT6 interacting proteins by both yeast-2-hybrid and co-immunoprecipitation studies. We report a number of interacting partners which strengthen previous findings that SIRT6 functions in base excision repair (BER), and novel interactors which suggest a role in nucleosome and chromatin remodeling, the cell cycle and NFκB biology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of ...Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is ...an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment.
The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE--the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface.
We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.
T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities ...typify adaptive immunity. Conversely, large compartments of γδTCR
intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (V
chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.
We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all ...SH‐groups modified by 5,5′‐dithiobis(2‐nitrobenzoate); (2) O‐R‐type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O‐P‐type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O‐R‐type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg‐ATP. Chemically modified GAPDH and the O‐R‐type mutant dimers inhibited GroEL‐assisted refolding of urea‐denatured wild‐type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O‐R‐dimers, the O‐P‐type mutant dimers neither bound nor affected GroEL‐assisted refolding of the wild‐type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.
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Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function ...SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10–100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.
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The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we ...wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.
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The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 ...inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Abstract
Background: Recent studies have highlighted the contribution of gamma delta (γδ) T cells in the immune response to cancer, and their infiltration in tumors is a positive prognostic factor. ...Vδ1 T cells are the most abundant γδ T cells in epithelial tissues including the gut and skin and form a significant proportion of tumour infiltrating lymphocytes (TILs) in a number of solid tumour indications. Tissue-resident cytotoxic Vδ1 γδ T cells are of particular interest in immunotherapy approaches for their role in recognising and eradicating cancer cells, providing new avenues for antibody tissue-based immunological intervention. Vδ1 γδ T cells combine features of innate and adaptive immunity: in contrast to αβ T cells that rely on antigen-presentation via MHC, γδ T cells recognize and kill transformed cells by direct recognition of a pattern of stress associated ligands expressed on their surface, opening up a potential range of cold tumours with decreased or impaired tumour antigen presentation. Adaptate’s first-in-class approach is to harness the specific anti-tumour properties of Vδ1 T cells using activating immunomodulatory antibodies.
Methods: Adaptate’s ADT-010 is a first-in-class, fully human, monoclonal antibody which selectively recognizes and binds to the Vδ1 TCR leading to Vδ1 T cell activation. ADT-010 was selected via high-stringency antigen binding using a human phage display library. To elucidate its mechanism of action, the binding and functional activity of ADT-010 was evaluated on expanded skin-derived Vδ1 T cells in vitro.
Results: ADT-010 specifically enhanced the Vδ1 killing of cancer cells whilst sparing healthy non-diseased cells. The functional activity of ADT-010 on expanded skin-derived Vδ1 T cells was investigated in the presence or absence of tumour cells. ADT-010 mediated activation of Vδ1 T cells in the presence of target tumour cells was demonstrated through TCR downregulation, CD107a and CD25 upregulation, and enhanced anti-tumour cytotoxicity. Taken together the data indicates that Vδ1 TCR engagement on γδ T cells by ADT-010 results in their full activation and enhanced cytotoxicity only in the presence of target tumour cells.
Conclusions: Despite recent advances in immune therapies in oncology, there currently remains a significant unmet need for effective and safe treatments for solid tumours. The challenges of treating such tumours which include infiltration into the tumour tissue and discrimination between healthy and tumour cells have the potential to be addressed by novel approaches such as Adaptate’s ADT-010 antibody. ADT-010 specifically activates a population of tumour-resident Vδ1 T cells that can then orchestrate a potent and highly selective anti-tumour response through direct killing.Adaptate’s first-in-class ADT-010 is being progressed to the clinic for the treatment of solid tumour indications.
Citation Format: Jose Munoz-Olaya, Aoife McGinley, Daniel Foxler, Samuel Samuel, Tanguy Lechertier, Shefali Bhumbra, Robert Good, Luke Cave, Oxana Polyakova, Mark Uden, Mihriban Tuna, Natalie Mount. ADT-010, a novel immunomodulatory antibody that harnesses the anti-tumor properties of tissue-resident Vδ1 γδ T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4254.
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