Elucidating the Molecular Mechanism of CYLD-Mediated Necrosis: A Dissertation

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation

CYLD deubiquitinates RIP1 in the TNFalpha-induced necrosome to facilitate kinase activation and programmed necrosis

BACKGROUND: Necroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFalpha is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that RIP1 is indeed the primary target of CYLD in TNFalpha-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD(-/-) cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD(-/-) cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD(-/-) cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD(-/-) cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation. CONCLUSIONS/SIGNIFICANCE: Our results show that CYLD regulates RIP1 ubiquitination in the TNFalpha-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly

Influenza Virus Infection Model With Density Dependence Supports Biphasic Viral Decay

Mathematical models that describe infection kinetics help elucidate the time scales, effectiveness, and mechanisms underlying viral growth and infection resolution. For influenza A virus (IAV) infections, the standard viral kinetic model has been used to investigate the effect of different IAV proteins, immune mechanisms, antiviral actions, and bacterial coinfection, among others. We sought to further define the kinetics of IAV infections by infecting mice with influenza A/PR8 and measuring viral loads with high frequency and precision over the course of infection. The data highlighted dynamics that were not previously noted, including viral titers that remain elevated for several days during mid-infection and a sharp 4–5 log10 decline in virus within 1 day as the infection resolves. The standard viral kinetic model, which has been widely used within the field, could not capture these dynamics. Thus, we developed a new model that could simultaneously quantify the different phases of viral growth and decay with high accuracy. The model suggests that the slow and fast phases of virus decay are due to the infected cell clearance rate changing as the density of infected cells changes. To characterize this model, we fit the model to the viral load data, examined the parameter behavior, and connected the results and parameters to linear regression estimates. The resulting parameters and model dynamics revealed that the rate of viral clearance during resolution occurs 25 times faster than the clearance during mid-infection and that small decreases to this rate can significantly prolong the infection. This likely reflects the high efficiency of the adaptive immune response. The new model provides a well-characterized representation of IAV infection dynamics, is useful for analyzing and interpreting viral load dynamics in the absence of immunological data, and gives further insight into the regulation of viral control

Dynamically linking influenza virus infection kinetics, lung injury, inflammation, and disease severity

Influenza viruses cause a significant amount of morbidity and mortality. Understanding host immune control efficacy and how different factors influence lung injury and disease severity are critical. We established and validated dynamical connections between viral loads, infected cells, CD

The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4+ T cells

Interleukin (IL) 6 is a proinflammtory cytokine produced by antigen-presenting cells and nonhematopoietic cells in response to external stimuli. It was initially identified as a B cell growth factor and inducer of plasma cell differentiation in vitro and plays an important role in antibody production and class switching in vivo. However, it is not clear whether IL-6 directly affects B cells or acts through other mechanisms. We show that IL-6 is sufficient and necessary to induce IL-21 production by naive and memory CD4+ T cells upon T cell receptor stimulation. IL-21 production by CD4+ T cells is required for IL-6 to promote B cell antibody production in vitro. Moreover, administration of IL-6 with inactive influenza virus enhances virus-specific antibody production, and importantly, this effect is dependent on IL-21. Thus, IL-6 promotes antibody production by promoting the B cell helper capabilities of CD4+ T cells through increased IL-21 production. IL-6 could therefore be a potential coadjuvant to enhance humoral immunity

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

CYLD does not regulate RIP1 ubiquitination within the TNFR1 complex.

<p>(<b>A</b>) Recruitment of CYLD and A20 to the TNFR-1 complex. TNFR1 complex was purified from TNF, zVAD and CHX treated cells by immunoprecipitation (IP). The recruitment of RIP1, CYLD and A20 was assessed via Western Blot. Control IPs with isotype-matched IgG were included to show the specificity of binding to TNFR-1. (<b>B</b>) CYLD^+/+ and CYLD^-/- MEFs were treated with TNF for the indicated times. Recruitment of polyubiquitinated RIP1 to TNFR-1 was determined by Western Blot. (<b>C</b>) IκBα phosphorylation and degradation was normal in CYLD^-/- MEFs.</p

Schematic diagram of the proposed mechanism of CYLD-mediated necrosis.

<p>Although CYLD is recruited to the TNFR-1 complex, it dose not deubiquitinate RIP1 within the this compartment. Upon transition to the cytosol, CYLD deubiquitinates RIP1 within the NP-40 insoluble fraction to actively promote necrosome phosphorylation and activation. In addition to regulating RIP1 ubiquitination in the necrosome, CYLD also controls downstream events of necrosis such as ROS production. </p

RIP1-dependent necrosis occurs in CYLD^-/- MEFs.

<p>Wild type (CYLD^+/+) and CYLD^-/- MEFs were treated with (<b>A</b>) TNF and zVAD, (<b>B</b>) TNF, Smac mimetic and zVAD, or (<b>C</b>) TNF, cycloheximide (CHX) and zVAD-fmk for 12 hours. Cell death was determined by staining with propidium iodide (PI) and analyzed via flow cytometry. The inset shows loss of CYLD expression in CYLD^-/- MEFs using a C-terminal specific CYLD antibody. In (<b>D</b>), cells were treated with 10 ng/ml TNF, CHX and zVAD-fmk and cell death was measured at the indicated times following treatment. Representative of three experiments. (<b>E</b>) Nec-1 inhibited TNF-induced necrosis in CYLD^-/- MEFs. Representative of two experiments. (<b>F</b>) CYLD^+/+ and CYLD^-/- MEFs were treated with TNF and CHX for 12 hours. Cell death was determined by PI staining and flow cytometry. Representative of three experiments. (<b>G</b>) MEFs were treated with 10 ng/ml TNF and CHX for the indicated times. Representative of two experiments. (<b>H</b>-<b>J</b>) MEFs were treated with (<b>H</b>) TRAIL, (<b>I</b>) FasL or (<b>J</b>) staurosporine (STS) for 12 hours. Cell death was determined by PI exclusion and flow cytometry.</p