Marc Schuster

Phosphorylation of Atg5 by the Gadd45β-MEKK4-p38 pathway inhibits autophagy.

Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45β)–MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45β-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45β/MEKK4 negatively regulates the autophagic process.

Autophagy in T-cell development, activation and differentiation.

Autophagy is a vital catabolic process for degrading bulky cytosolic contents, which cannot be resorbed via the proteasome. First described as a survival mechanism during nutrient starvation conditions, recent reports have demonstrated that autophagy supports metabolic functions of T cells at various stages of maturation and effector function. Autophagy is crucial for T‐cell development at the precursor stage as self‐renewability and quiescence of hematopoietic stem cells depend on autophagy of the mitochondria and the endoplasmic reticulum. Later, during development in the thymus, autophagy regulates peptide presentation in stromal cells and professional antigen‐presenting cells, which mediate thymocyte selection. Furthermore, the metabolic changes when mature T cells enter the periphery and when they are activated are both dependent on autophagy. Lastly, autophagy prevents early aging and, thus, ensures maintenance of memory T cells.

Atypical IκB proteins – nuclear modulators of NF-κB signaling

Nuclear factor κB (NF-κB) controls a multitude of physiological processes such as cell differentiation, cytokine expression, survival and proliferation. Since NF-κB governs embryogenesis, tissue homeostasis and the functions of innate and adaptive immune cells it represents one of the most important and versatile signaling networks known. Its activity is regulated via the inhibitors of NF-κB signaling, the IκB proteins. Classical IκBs, like the prototypical protein IκBα, sequester NF-κB transcription factors in the cytoplasm by masking of their nuclear localization signals (NLS). Thus, binding of NF-κB to the DNA is inhibited. The accessibility of the NLS is controlled via the degradation of IκBα. Phosphorylation of the conserved serine residues 32 and 36 leads to polyubiquitination and subsequent proteasomal degradation. This process marks the central event of canonical NF-κB activation. Once their NLS is accessible, NF-κB transcription factors translocate into the nucleus, bind to the DNA and regulate the transcription of their respective target genes. Several studies described a distinct group of atypical IκB proteins, referred to as the BCL-3 subfamily. Those atypical IκBs show entirely different sub-cellular localizations, activation kinetics and an unexpected functional diversity. First of all, their interaction with NF-κB transcription factors takes place in the nucleus in contrast to classical IκBs, whose binding to NF-κB predominantly occurs in the cytoplasm. Secondly, atypical IκBs are strongly induced after NF-κB activation, for example by LPS and IL-1β stimulation or triggering of B cell and T cell antigen receptors, but are not degraded in the first place like their conventional relatives. Finally, the interaction of atypical IκBs with DNA-associated NF-κB transcription factors can further enhance or diminish their transcriptional activity. Thus, they do not exclusively act as inhibitors of NF-κB activity. The capacity to modulate NF-κB transcription either positively or negatively, represents their most important and unique mechanistic difference to classical IκBs. Several reports revealed the importance of atypical IκB proteins for immune homeostasis and the severe consequences following their loss of function. This review summarizes insights into the physiological processes regulated by this protein class and the relevance of atypical IκB functioning.

IκBNS Regulates Murine Th17 Differentiation during Gut Inflammation and Infection

IL-17–producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A–producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS−/− Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS−/− mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections.

Atypical IκB proteins in immune cell differentiation and function.

The NF-κB/Rel signalling pathway plays a crucial role in numerous biological processes, including innate and adaptive immunity. NF-κB is a family of transcription factors, whose activity is regulated by the inhibitors of NF-κB (IκB). The IκB proteins comprise two distinct groups, the classical (cytoplasmic) and the atypical (nuclear) IκB proteins. Although the cytoplasmic regulation of NF-κB is well characterised, its nuclear regulation mechanisms remain marginally elucidated. However, work from recent years indicated that nuclear IκBs contribute significantly to the modulation of NF-κB-mediated transcription in the immune system. Here, we discuss the role of the atypical IκB proteins Bcl-3, IκBζ, IκBNS, IκBη and IκBL for the regulation of gene expression and effector functions in immune cells.

The role of c-FLIP splice variants in urothelial tumours

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkins lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIPlong (c-FLIPL) and c-FLIPshort (c-FLIPS), which can have opposing functions. We observed diminished expression of the c-FLIPL isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIPS was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIPL to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIPL isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.

SLy2 targets the nuclear SAP30/HDAC1 complex

The adapter protein SLy2 (SH3 protein expressed in lymphocytes 2), also named HACS1, NASH1 or SAMSN1, is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells and myelomas. Endogenous SLy2 expression was shown to be upregulated in primary B cells upon differentiation and proliferation-inducing stimuli, and transduction experiments suggest a stimulatory role for SLy2 in B cell differentiation to plasma cells. However the signalling pathways regulated by SLy2 remain unknown. In this study we identify novel interaction partners of SLy2 providing a molecular framework for its function. We show that phosphorylated SLy2 directly interacts with 14-3-3 proteins via a previously unrecognized phosphorylation site. Furthermore, we demonstrate that 14-3-3 proteins control nucleo-cytoplasmic shuttling of SLy2 by retaining phosphorylated SLy2 in the cytoplasm. In the nucleus, SLy2 interacts with the SAP30/HDAC1 complex and regulates the activity of HDAC1. Thus, our findings unravel a novel mechanism how SLy2 localization is controlled and implicate SLy2 in the epigenetic control of gene expression.

Cellular-FLIP, Raji isoform (c-FLIP R ) modulates cell death induction upon T-cell activation and infection

Dysregulation of apoptosis caused by an imbalance of pro‐ and anti‐apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular‐FLIP (c‐FLIP) proteins inhibit apoptosis directly at the death‐inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c‐FLIPL and c‐FLIPS are well characterized, the function of c‐FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c‐FLIPR in activated lymphocytes for the first time. To analyze c‐FLIPR function in vivo, we generated transgenic mice expressing murine c‐FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c‐FLIPR transgenic mice were protected against CD95‐induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T‐cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c‐FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild‐type mice. We conclude that c‐FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

c-REL and IκB NS Govern Common and Independent Steps of Regulatory T Cell Development from Novel CD122-Expressing Pre-Precursors

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκBNS are important regulators of Foxp3 induction in Treg precursors upon γ-chain cytokine stimulation. In c-REL/IκBNS double-deficient mice, Treg numbers were dramatically reduced, indicating that together, c-REL and IκBNS are pivotal for Treg development. However, despite the highly reduced Treg compartment, double-deficient mice did not develop autoimmunity even when aged to more than 1 y, suggesting that c-REL and IκBNS are required for T cell effector function as well. Analyzing Treg development in more detail, we identified a CD122+ subset within the CD25−Foxp3− precursor population, which gave rise to classical CD25+Foxp3− Treg precursors. Importantly, c-REL, but not IκBNS, controlled the generation of classical CD25+Foxp3− precursors via direct binding to the Cd25 locus. Thus, we propose that CD4+GITR+CD122+CD25−Foxp3− cells represent a Treg pre-precursor population, whose transition into Treg precursors is mediated via c-REL.