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    Ang II Promotes Cardiac Autophagy and Hypertrophy via Orai1/STIM1.
    Zheng, C-B ; Gao, W-C ; Xie, M ; Li, Z ; Ma, X ; Song, W ; Luo, D ; Huang, Y ; Yang, J ; Zhang, P ; Huang, Y ; Yang, W ; Yao, X (Frontiers Media S.A., 2021)
    The pathophysiology of cardiac hypertrophy is complex and multifactorial. Both the store-operated Ca2+ entry (SOCE) and excessive autophagy are the major causative factors for pathological cardiac hypertrophy. However, it is unclear whether these two causative factors are interdependent. In this study, we examined the functional role of SOCE and Orai1 in angiotensin II (Ang II)-induced autophagy and hypertrophy using in vitro neonatal rat cardiomyocytes (NRCMs) and in vivo mouse model, respectively. We show that YM-58483 or SKF-96365 mediated pharmacological inhibition of SOCE, or silencing of Orai1 with Orail-siRNA inhibited Ang II-induced cardiomyocyte autophagy both in vitro and in vivo. Also, the knockdown of Orai1 attenuated Ang II-induced pathological cardiac hypertrophy. Together, these data suggest that Ang II promotes excessive cardiomyocyte autophagy through SOCE/Orai1 which can be the prime contributing factors in cardiac hypertrophy.
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    Trajectory of immune evasion and cancer progression in hepatocellular carcinoma.
    Nguyen, PHD ; Wasser, M ; Tan, CT ; Lim, CJ ; Lai, HLH ; Seow, JJW ; DasGupta, R ; Phua, CZJ ; Ma, S ; Yang, J ; Suthen, SDO ; Tam, WL ; Lim, TKH ; Yeong, J ; Leow, WQ ; Pang, YH ; Soon, G ; Loh, TJ ; Wan, WK ; Chan, CY ; Cheow, PC ; Toh, HC ; Kow, A ; Dan, YY ; Kam, JH ; Iyer, S ; Madhavan, K ; Chung, A ; Bonney, GK ; Goh, BKP ; Fu, N ; Yu, VC ; Zhai, W ; Albani, S ; Chow, PKH ; Chew, V (Nature Portfolio, 2022-03-17)
    Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.
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    Caspase-2 does not play a critical role in cell death induction and bacterial clearance during Salmonella infection
    Engel, S ; Doerflinger, M ; Lee, AR ; Strasser, A ; Herold, MJ ; Bedoui, S ; Bachem, A (Springer Nature, 2021-12)
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    Emerging connectivity of programmed cell death pathways and its physiological implications
    Bedoui, S ; Herold, MJ ; Strasser, A (Nature Research, 2020-11)
    The removal of functionally dispensable, infected or potentially neoplastic cells is driven by programmed cell death (PCD) pathways, highlighting their important roles in homeostasis, host defence against pathogens, cancer and a range of other pathologies. Several types of PCD pathways have been described, including apoptosis, necroptosis and pyroptosis; they employ distinct molecular and cellular processes and differ in their outcomes, such as the capacity to trigger inflammatory responses. Recent genetic and biochemical studies have revealed remarkable flexibility in the use of these PCD pathways and indicate a considerable degree of plasticity in their molecular regulation; for example, despite having a primary role in inducing pyroptosis, inflammatory caspases can also induce apoptosis, and conversely, apoptotic stimuli can trigger pyroptosis. Intriguingly, this flexibility is most pronounced in cellular responses to infection, while apoptosis is the dominant cell death process through which organisms prevent the development of cancer. In this Review, we summarize the mechanisms of the different types of PCD and describe the physiological and pathological processes that engage crosstalk between these pathways, focusing on infections and cancer. We discuss the intriguing notion that the different types of PCD could be seen as a single, coordinated cell death system, in which the individual pathways are highly interconnected and can flexibly compensate for one another.
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    Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.
    Kato, Y ; Steiner, TM ; Park, H-Y ; Hitchcock, RO ; Zaid, A ; Hor, JL ; Devi, S ; Davey, GM ; Vremec, D ; Tullett, KM ; Tan, PS ; Ahmet, F ; Mueller, SN ; Alonso, S ; Tarlinton, DM ; Ploegh, HL ; Kaisho, T ; Beattie, L ; Manton, JH ; Fernandez-Ruiz, D ; Shortman, K ; Lahoud, MH ; Heath, WR ; Caminschi, I (American Association of Immunologists, 2020-10-01)
    Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.
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    HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells
    Kueh, AJ ; Eccles, S ; Tang, L ; Garnham, AL ; May, RE ; Herold, MJ ; Smyth, GK ; Voss, AK ; Thomas, T (American Society for Microbiology, 2020-02-01)
    HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.
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    Location, location, location! Rewiring IL-2 circuits defines context-specific outcomes
    Rankin, L ; Gray, D (WILEY, 2022-09)
    Here, we highlight a recent publication by Whyte et al. that reveals diverse immune outcomes of interleukin (IL)-2 expression in distinct microenvironments. Their definition of context-dependent IL-2 networks paves the way for the development of tissue-specific therapies that enlist the potent immuno-modulatory activity of IL-2.
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    HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis
    Freeman, AJ ; Vervoort, SJ ; Michie, J ; Ramsbottom, KM ; Silke, J ; Kearney, CJ ; Oliaro, J (WILEY, 2021-11-04)
    The success of cancer immunotherapy is limited to a subset of patients, highlighting the need to identify the processes by which tumors evade immunity. Using CRISPR/Cas9 screening, we reveal that melanoma cells lacking HOIP, the catalytic subunit of LUBAC, are highly susceptible to both NK and CD8+ T-cell-mediated killing. We demonstrate that HOIP-deficient tumor cells exhibit increased sensitivity to the combined effect of the inflammatory cytokines, TNF and IFN-γ, released by NK and CD8+ T cells upon target recognition. Both genetic deletion and pharmacological inhibition of HOIP augment tumor cell sensitivity to combined TNF and IFN-γ. Together, we unveil a protective regulatory axis, involving HOIP, which limits a transcription-dependent form of cell death that engages both intrinsic and extrinsic apoptotic machinery upon exposure to TNF and IFN-γ. Our findings highlight HOIP inhibition as a potential strategy to harness and enhance the killing capacity of TNF and IFN-γ during immunotherapy.
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    The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK
    Ke, FS ; Holloway, S ; Uren, RT ; Wong, AW ; Little, MH ; Kluck, RM ; Voss, AK ; Strasser, A (WILEY, 2022-08-01)
    The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok-/- Bax-/- Bak-/- triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal. This raises the question if all apoptosis had been abolished in the TKO mice or if other BCL-2 family members could act as effectors of apoptosis. Here, we investigated the role of BID, generally considered to link the extrinsic and intrinsic apoptosis pathways, acting as a BH3-only protein initiating apoptosis upstream of BAX and BAK. We found that Bok-/- Bax-/- Bak-/- Bid-/- quadruple knockout (QKO) mice have additional developmental anomalies compared to TKO mice, consistent with a role of BID, not only upstream but also in parallel to BAX, BAK and BOK. Mitochondrial experiments identified a small cytochrome c-releasing activity of full-length BID. Collectively, these findings suggest a new effector role for BID in the intrinsic apoptosis pathway.
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    Dynamic reconfiguration of pro-apoptotic BAK on membranes
    Sandow, JJ ; Tan, IK ; Huang, AS ; Masaldan, S ; Bernardini, JP ; Wardak, AZ ; Birkinshaw, RW ; Ninnis, RL ; Liu, Z ; Dalseno, D ; Lio, D ; Infusini, G ; Czabotar, PE ; Webb, A ; Dewson, G (WILEY, 2021-10-18)
    BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.