USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy.

ULK1 (unc-51 like autophagy activating kinase 1) is a core component at multiple steps of canonical macroautophagy/autophagy. The activity of ULK1 is tightly regulated by several post-translational modifications, including ubiquitination, yet the deubiquitinase (DUB) responsible for its reversible deubiquitination has not been described. Here, we identified USP1 (ubiquitin specific peptidase 1) as a key player in the modulation of ULK1 K63-linked deubiquitination.

Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin.

CAPNS1 is essential for stability and function of the ubiquitous calcium-dependent proteases micro- and milli-calpain. Upon inhibition of the endoplasmic reticulum Ca ATPase by 100 nM thapsigargin, both micro-calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1-dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1-containing vesicles with LC3 bodies in control cells.

The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways.

Calpain restrains the stem cells compartment in breast cancer.

CAPNS1 is essential for the stability and function of ubiquitous CAPN1 and CAPN2. Calpain modulates by proteolytic cleavage many cellular substrates and its activity is often deregulated in cancer cells, therefore calpain inhibition has been proposed as a therapeutical strategy for a number of malignancies. Here we show that CAPNS1 depletion is coupled to impairment of MCF7 and MCF10AT cell lines growth on plate and defective architecture of mammary acini derived from MCF10A cells.

CAPNS1 regulates USP1 stability and maintenance of genome integrity.

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines.

Beside the MEF2 axis: unconventional functions of HDAC4.

The class IIa deacetylase HDAC4 is unequivocally known as a negative regulator of MEF2-dependent transcription. In the past years several works have allowed us to understand how different signals, mirroring specific environmental circumstances keep in check the repressive action of HDAC4 against MEF2s. At the same time, pieces of evidence have gradually accumulated about HDAC4 activities emancipated from MEF2s.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

DNA damage response links calpain to cellular senescence.

Senescence represents an important barrier against cellular transformation. Here we show that CAPNS1 depletion impairs senescence induction both in BJ-ET H-Ras(v12) inducible human fibroblasts upon Ras induction and in HT1080 targeted to senescence by treatment with low doses of doxorubicin. We further show that CAPNS1 depletion is coupled to reduced levels of H2AX phosphorylation, not only in Ras(v12) induced BJ-ET fibroblasts, but also in a number of cellular systems upon genotoxic stress.

p65/RelA binds and activates the beclin 1 promoter.

We unveiled novel p65/RelA consensus sites in the promoter of the beclin 1 gene and demonstrate that p65/RelA positively modulates canonical autophagy in various human cell lines both under basal conditions and upon induction by ceramide. Interestingly, we find that T cell receptor-dependent activation of Jurkat cells triggers an increase in the binding of p65/RelA to the beclin 1 promoter accompanied by enhanced autophagy, suggesting that p65/RelA could regulate T-cell activation and homeostasis through autophagy.

p65/RelA modulates BECN1 transcription and autophagy.

Recently, autophagy has emerged as a critical process in the control of T-cell homeostasis. Given the pivotal role of NF-kappaB in the signaling events of T cells, we have analyzed and unveiled a conserved NF-kappaB binding site in the promoter of the murine and human BECN1 autophagic gene (Atg6). Accordingly, we demonstrate that the NF-kappaB family member p65/RelA upregulates BECN1 mRNA and protein levels in different cellular systems.

Inhibitors of the ubiquitin-proteasome system are not all alike: identification of a new necrotic pathway.

In response to stress, cells can not only activate survival pathways, but also death pathways, when the stresses are ungovernable. The case of proteasome impairment is emblematic in this respect. Autophagy is induced, as a prosurvival response, which antagonizes apoptosis.

The Isopeptidase Inhibitor G5 Triggers a Caspase-independent Necrotic Death in Cells Resistant to Apoptosis: A COMPARATIVE STUDY WITH THE PROTEASOME INHIBITOR BORTEZOMIB.

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO).

Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A.

Here, we show that FoxO3A transcription factor is upregulated upon calpain small-1 (CAPNS1) depletion both in mouse embryonic fibroblasts (MEFs) and in the human mammary carcinoma cell line MCF-7. On starvation, CAPNS1 depletion is associated with a higher rate of FoxO3A dephosphorylation and translocation to the nucleus and to a sharper increase in the levels of p27Kip1 and Bim, the products of two FoxO target genes. Notably, FoxO3A depletion in CAPNS1-/- MEFs reduces both the induction of Bim and apoptosis.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.

The calpain system as a modulator of stress/damage response.

Ubiquitously expressed mu- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene. The mu- and m-calpain proteases have been implicated in both pro-or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells.

Calpain as a novel regulator of autophagosome formation.

Ubiquitously expressed micro- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene. The micro- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells.

Gene expression profile of primary gastric cancer: towards the prediction of lymph node status.

Background: The identification of gastric tumors associated with a higher risk of lymph node metastasis could help surgeons select patients who may benefit from extended lymph node dissection. The aim of this study was to screen the genome in the search of primary gastric cancer gene expression profiles that might predict lymph node status.

Methods: The gene expression profile was evaluated in frozen tumor samples obtained from 32 patients with primary gastric adenocarcinomas.

Calpain is required for macroautophagy in mammalian cells.

Ubiquitously expressed micro- and millicalpain, which both require the calpain small 1 (CAPNS1) regulatory subunit for function, play important roles in numerous biological and pathological phenomena. We have previously shown that the product of GAS2, a gene specifically induced at growth arrest, is an inhibitor of millicalpain and that its overexpression sensitizes cells to apoptosis in a p53-dependent manner (Benetti, R., G.

Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies.

The promising effects of the proteasome inhibitor bortezomib (Velcade, PS-341) in the treatment of certain types of cancer have fired up the interest on this multicatalytic proteolytic machinery. A number of recent reviews thoroughly describe various aspects of the ubiquitin-proteasome system and its importance in the control of cell growth and tumorigenesis. Here, we will focus on recent data unveiling a link between the proteasome and some elements of the apoptotic machinery including Bcl-2 members, caspases, IAPs and IAP antagonists.

Ceramide triggers an NF-kappaB-dependent survival pathway through calpain.

We have shown that C2 ceramide, a cell-permeable analog of this lipid second messenger, triggers an NF-kappaB dependent survival pathway that counteracts cell death. Activation of NF-kappaB and subsequent induction of prosurvival genes relies on calpain activity and is prevented on silencing of the calpain small subunit (Capn4) that is required for the function of ubiquitous calpains. We have demonstrated that p105 (NF-kappaB1) and its proteolytic product p50 can be targets of micro- and milli-calpain in vitro and that a p50 deletion mutant, lacking both the N- and the C-terminal ends, is resistant to calpain-mediated degradation.

Human synaptobrevin-like 1 gene basal transcription is regulated through the interaction of selenocysteine tRNA gene transcription activating factor-zinc finger 143 factors with evolutionary conserved cis-elements.

The synaptobrevin-like 1 (SYBL1) gene is ubiquitously expressed and codes for an unusual member of the v-SNAREs molecules implicated in cellular exocytosis. This X-linked gene has the peculiarity of also being present on the Y chromosome in a transcriptional inactive status. Moreover, although ubiquitous, the function of SYBL1 is prominent in specific tissues, such as brain.

Glycogen synthase kinase-3 beta regulates NF-kappa B1/p105 stability.

A number of different kinases have been implicated in NF-kappa B regulation and survival function. Here we investigated the molecular cross-talk between glycogen synthase kinase-3 beta (GSK-3 beta) and the p105 precursor of the NF-kappa B p50 subunit. GSK-3 beta forms an in vivo complex with and specifically phosphorylates NF-kappa B1/p105 at Ser-903 and Ser-907 in vitro.

Gas6 anti-apoptotic signaling requires NF-kappa B activation.

The growth arrest-specific 6 gene product Gas6 is a growth and survival factor related to protein S. Gas6 is the ligand of Axl receptor tyrosine kinase; upon binding to its receptor Gas6 activates the phosphatidylinositol 3-OH kinase (PI3K) and its downstream targets S6K and Akt. Gas6 anti-apoptotic signaling was previously shown to require functional PI3K and Akt and to involve Bad phosphorylation in serum-starved NIH 3T3 cells.