SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.Peer reviewe

Caspase-8 inhibition represses initial human monocyte activation in septic shock model

In septic patients, the onset of septic shock occurs due to the over-activation of monocytes. We tested the therapeutic potential of directly targeting innate immune cell activation to limit the cytokine storm and downstream phases. We initially investigated whether caspase-8 could be an appropriate target given it has recently been shown to be involved in microglial activation. We found that LPS caused a mild increase in caspase-8 activity and that the caspase-8 inhibitor IETD-fmk partially decreased monocyte activation. Furthermore, caspase-8 inhibition induced necroptotic cell death of activated monocytes. Despite inducing necroptosis, caspase-8 inhibition reduced LPS-induced expression and release of IL-1β and IL-10. Thus, blocking monocyte activation has positive effects on both the pro and anti-inflammatory phases of septic shock. We also found that in primary mouse monocytes, caspase-8 inhibition did not reduce LPS-induced activation or induce necroptosis. On the other hand, broad caspase inhibitors, which have already been shown to improve survival in mouse models of sepsis, achieved both. Thus, given that monocyte activation can be regulated in humans via the inhibition of a single caspase, we propose that the therapeutic use of caspase-8 inhibitors could represent a more selective alternative that blocks both phases of septic shock at the source.Unión Europea, Ministerio de Economía y Competitividad SAF2012-39029Unión Europea, Ministerio de Economía y Competitividad SAF2015-64171REspaña,Junta de Andalucía P10-CTS-649

The many faces of p57Kip2 : Acting in apoptosis, differentiation and cytoskeleton reorganisation

p57Kip2 is a known cyclin dependent kinase inhibitor, which has been suggested to be a tumor suppressor gene. Indeed various human cancers show a reduction in p57Kip2 expression, indicating that this protein might be of relevance in tumorigenesis. Mutated forms of p57Kip2 have rarely been detected in human tumors; rather epigenetic mechanisms are involved in inactivating the expression of the gene. Inactivation of the p57 Kip2 gene correlates with disease progression and poor prognosis for the patient, indicating that reactivation of p57Kip2 in cancer cells could possible inhibit tumor cell growth. Prior research has primarily focused on p57Kip2´s role as cell-cycle regulator, whereas the aim of this thesis is to study its function during other cellular events such as apoptosis, cytoskeleton reorganisation and differentiation of neuronal cells. We report that selective expression of p57Kip2 sensitizes tumor cells to cell death induced by different drugs. This function is independent of its role as a cyclin dependent kinase inhibitor. Our studies also show that p57Kip2 primarily promotes the mitochondrial apoptotic pathway. In accordance, we found that Bcl-2 overexpression or VDAC inhibition were able to inhibit p57Kip2 cell death promoting effect. We also reveal that p57Kip2 is a direct target gene for p73β, which is known for its pro-apoptotic properties. Moreover, the p73β-induced p57Kip2 expression contributes to mitochondrial events related to apoptotic cell death. Furthermore, we establish that p57Kip2 expression promotes actin stress fiber formation in cells. It interacts with, and activates the actin cytoskeleton modifying enzyme, LIM-Kinase-1. This activation resulted into increase phosphorylation, inactivation of cofilin and in a reduction of actin protein mobile fraction, ultimately, affecting negatively cell mobility. Remodelling of actin cytoskeleton plays a key role in cell migration and has implication for invasion and metastasis, supporting the proposal that p57Kip2 can act as a tumor suppressor gene by affecting the cytoskeleton. We further investigated the role of p57Kip2 in neural stem cell differentiation. We show that p57Kip2 transiently accumulates in the nuclei of neural progenitors during early astrocyte differentiation and represses neuronal differentiation. It can interact with a subset of pro-neuronal bHLH factors, including Mash1. p57Kip2 inhibits the transcriptional activity of Mash1 and thereby represses neuronal differentiation, possibly to allow proper glial differentiation. These findings once again confirm the importance of p57Kip2 during development and differentiation. In conclusion, our work shows that p57Kip2 plays essential roles during several cellular processes besides acting as a cell-cycle regulator. This thesis contributes to the understanding of p57Kip2 s role in apoptosis, differentiation and cytoskeleton reorganisation

Protein Kinase C-Dependent Phosphorylation Regulates the Cell Cycle-Inhibitory Function of the p73 Carboxy Terminus Transactivation Domain▿ †

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. However, the regulatory mechanisms controlling the distinct roles for p73 in these two processes have remained unclear. Here, we report that p73 is able to induce cell cycle arrest independently of its amino-terminal transactivation domain, whereas this domain is crucial for p73 proapoptotic functions. We also characterized a second transactivation domain in the carboxy terminus of p73 within amino acid residues 381 to 399. This carboxy terminus transactivation domain was found to preferentially regulate genes involved in cell cycle progression. Moreover, its activity is regulated throughout the cell cycle and modified by protein kinase C-dependent phosphorylation at serine residue 388. Our results suggest that this novel posttranslational modification within the p73 carboxy terminus transactivation domain is involved in the context-specific guidance of p73 toward the selective induction of cell cycle arrest

SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12

Inactivating mutations in the SETD2 gene, encoding for a nonredundant histone H3 methyltransferase and regulator of transcription, is a frequent molecular feature in clear cell renal cell carcinomas (ccRCC). SETD2 deficiency is associated with recurrence of ccRCC and bears low prognostic values. Targeting autophagy, a conserved catabolic process with critical functions in maintenance of cellular homeostasis and cell conservation under stress condition, is emerging as a potential therapeutic strategy to combat ccRCC. Epigenetics-based pathways are now appreciated as key components in the regulation of autophagy. However, whether loss of function in the SETD2 histone modifying enzyme occurring in ccRCC cells may impact on their ability to undergo autophagy remained to be explored. Here, we report that SETD2 deficiency in RCC cells is associated with the aberrant accumulation of both free ATG12 and of an additional ATG12-containing complex, distinct from the ATG5-ATG12 complex. Rescue of SETD2 functions in the SETD2 deficiency in RCC cells, or reduction of SETD2 expression level in RCC cells wild type for this enzyme, demonstrates that SETD2 deficiency in RCC is directly involved in the acquisition of these alterations in the autophagic process. Furthermore, we revealed that deficiency in SETD2, known regulator of alternative splicing, is associated with increased expression of a short ATG12 spliced isoform at the depend of the canonical long ATG12 isoform in RCC cells. The defect in the ATG12-dependent conjugation system was found to be associated with a decrease autophagic flux, in accord with the role for this ubiquitin-like protein conjugation system in autophagosome formation and expansion. Finally, we report that SETD2 and ATG12 gene expression levels are associated with favorable respective unfavorable prognosis in ccRCC patients. Collectively, our findings bring further argument for considering the SETD2 gene status of ccRCC tumors, when therapeutic interventions, such as targeting the autophagic process, are considered to combat these kidney cancers

Cell Death Dis

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms