Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses

Peri-substituted phosphorus-tellurium systems – an experimental and theoretical investigation of the P∙∙∙Te through-space interaction

The authors are thankful to the EPSRC, the EPSRC National Mass Spectrometry Service Centre (NMSSC) Swansea, the School of Chemistry St. Andrews, and EaStCHEM for support.A series of peri-substituted phosphorus-tellurium systems R’Te–Acenap–PR2 (R’ = Ph, p-An, Nap, Mes, Tip; R = iPr, Ph) exhibiting large “through space” spin-spin coupling constants and the “onset” of three-centre four-electron type interactions are presented. The influence of the substituents at the phosphorus and tellurium atoms as well as their behavior upon oxidation (with S, Se) or metal-coordination (Pt, Au) is discussed using NMR spectroscopy, single crystal X-ray diffraction, and advanced DFT studies including NBO, AIM and ELI-D analyses.PostprintPeer reviewe

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

Ubiquitination and phosphorylation of the CARD11-BCL10-MALT1 signalosome in T cells

Antigen receptor-induced signaling plays an important role in inflammation and immunity. Formation of a CARD11-BCL10-MALT1 (CBM) signaling complex is a key event in T- and B cell receptor-induced gene expression by regulating NF-κB activation and mRNA stability. Deregulated CARD11, BCL10 or MALT1 expression or CBM signaling have been associated with immunodeficiency, autoimmunity and cancer, indicating that CBM formation and function have to be tightly regulated. Over the past years great progress has been made in deciphering the molecular mechanisms of assembly and disassembly of the CBM complex. In this context, several posttranslational modifications play an indispensable role in regulating CBM function and downstream signal transduction. In this review we summarize how the different CBM components as well as their interplay are regulated by protein ubiquitination and phosphorylation in the context of T cell receptor signaling

CYLD, A20 and OTULIN deubiquitinases in NF-κB signaling and cell death : so similar, yet so different

Polyubiquitination of proteins has a pivotal role in the regulation of numerous cellular functions such as protein degradation, DNA repair and cell signaling. As deregulation of these processes can result in pathological conditions such as inflammatory diseases, neurodegeneration or cancer, tight regulation of the ubiquitin system is of tremendous importance. Ubiquitination by E3 ubiquitin ligases can be counteracted by the activity of several deubiquitinating enzymes (DUBs). CYLD, A20 and OTULIN have been implicated as key DUBs in the negative regulation of NF-kappa B transcription factor-mediated gene expression upon stimulation of cytokine receptors, antigen receptors and pattern recognition receptors, by removing distinct types of polyubiquitin chains from specific NF-kappa B signaling proteins. In addition, they control TNF-induced cell death signaling leading to apoptosis and necroptosis via similar mechanisms. In the case of A20, also catalytic-independent mechanisms of action have been demonstrated to have an important role. CYLD, A20 and OTULIN have largely overlapping substrates, suggesting at least partially redundant functions. However, mice deficient in one of the three DUBs show significant phenotypic differences, indicating also non-redundant functions. Here we discuss the activity and polyubiquitin chain-type specificity of CYLD, A20 and OTULIN, their specific role in NF-kappa B signaling and cell death, the molecular mechanisms that regulate their activity, their role in immune homeostasis and the association of defects in their activity with inflammation, autoimmunity and cancer

Importance of Validating Antibodies and Small Compound Inhibitors Using Genetic Knockout Studies—T Cell Receptor-Induced CYLD Phosphorylation by IKKε/TBK1 as a Case Study

CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it could still be prevented by MRT67307, indicating that the initially observed inhibitory effect of MRT67307 on TCR-induced CYLD phosphorylation is IKKε/TBK1-independent. Most surprisingly, the phospho(Ser418)-CYLD immunoreactive band was still detectable upon immunoblotting of cell extracts obtained from CYLD deficient cells. These data demonstrate the non-specificity of MRT67307 and phospho(Ser418)-CYLD specific antibodies, implying that previously published results based on these tools may also have led to wrong conclusions. We therefore advise to use genetic knockout studies or alternative approaches for a better validation of antibodies and small compound inhibitors. Interestingly, immunoprecipitation with the phospho(Ser418)-CYLD antibody, followed by immunoblotting with anti-CYLD, revealed that CYLD is phosphorylated by IKKε/TBK1 at Ser418 upon T cell stimulation, but that its direct detection with the phospho(Ser418)-CYLD-specific antibody in a western blot is masked by another inducible protein of the same size that is recognized by the same antibody

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<p>CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it could still be prevented by MRT67307, indicating that the initially observed inhibitory effect of MRT67307 on TCR-induced CYLD phosphorylation is IKKε/TBK1-independent. Most surprisingly, the phospho(Ser418)-CYLD immunoreactive band was still detectable upon immunoblotting of cell extracts obtained from CYLD deficient cells. These data demonstrate the non-specificity of MRT67307 and phospho(Ser418)-CYLD specific antibodies, implying that previously published results based on these tools may also have led to wrong conclusions. We therefore advise to use genetic knockout studies or alternative approaches for a better validation of antibodies and small compound inhibitors. Interestingly, immunoprecipitation with the phospho(Ser418)-CYLD antibody, followed by immunoblotting with anti-CYLD, revealed that CYLD is phosphorylated by IKKε/TBK1 at Ser418 upon T cell stimulation, but that its direct detection with the phospho(Ser418)-CYLD-specific antibody in a western blot is masked by another inducible protein of the same size that is recognized by the same antibody.</p

Defining the combinatorial space of PKC::CARD-CC signal transduction nodes.

peer reviewedSignal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling