Pasquale Vito

Participation of Presenilin 2 in Apoptosis: Enhanced Basal Activity Conferred by an Alzheimer Mutation

Overexpression of the familial Alzheimers disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or β-amyloid. Transfection of antisense PS2 conferred protection against apoptosis induced by trophic withdrawal in nerve growth factor-differentiated or amyloid precursor protein-expressing PC12 cells. The apoptotic cell death induced by PS2 protein was sensitive to pertussis toxin, suggesting that heterotrimeric GTP-binding proteins are involved. A PS2 mutation associated with familial Alzheimers disease was found to generate a molecule with enhanced basal apoptotic activity. This gain of function might accelerate the process of neurodegeneration that occurs in Alzheimers disease, leading to the earlier age of onset characteristic of familial Alzheimers disease.

Interfering with Apoptosis: Ca2+-Binding Protein ALG-2 and Alzheimer's Disease Gene ALG-3

Two apoptosis-linked genes, named ALG-2 and ALG-3, were identified by means of a functional selection strategy. ALG-2 codes for a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death. ALG-3, a partial complementary DNA that is homologous to the familial Alzheimers disease gene STM2, rescues a T cell hybridoma from T cell receptor- and Fas-induced apoptosis. These findings suggest that ALG-2 may mediate Ca2+-regulated signals along the death pathway and that cell death may play a role in Alzheimers disease.

ABIN-1 Binds to NEMO/IKKγ and Co-operates with A20 in Inhibiting NF-κB

Nuclear factor κB (NF-κB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-κB is dependent on the phosphorylation of the inhibitory subunit IκBα that is mediated by a multimeric, high molecular weight complex, called IκB kinase (IKK) complex. This is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO/IKKγ. The latter protein is essential for the activation of IKKs and NF-κB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-κB) as a NEMO/IKKγ-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-κB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-κB at the level of the IKK complex and that A20 inhibits activation of NF-κB by de-ubiquitination of NEMO/IKKγ. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKγ and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-κB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKγ and facilitates A20-mediated de-ubiquitination of NEMO/IKKγ, thus resulting in inhibition of NF-κB.

Requirement of the Familial Alzheimer's Disease Gene PS2 for Apoptosis OPPOSING EFFECT OF ALG-3

ALG-3, a truncated mouse homologue of the chromosome 1 familial Alzheimers disease gene PS2, rescues T hybridoma 3DO cells from T-cell receptor-induced apoptosis by inhibiting Fas ligand induction and Fas signaling. Here we show that ALG-3 transfected 3DO cells express a COOH-terminal PS2 polypeptide. Overexpression of PS2 in ALG-3 transfected 3DO cells reconstitutes sensitivity to receptor-induced cell death, suggesting that the artificial PS2 polypeptide functions as a dominant negative mutant of PS2. ALG-3 and antisense PS2 protect PC12 cells from glutamate-induced apoptosis but not from death induced by hydrogen peroxide or the free radical MPP+. Thus, the PS2 gene is required for some forms of cell death in diverse cell types, and its function is opposed by ALG-3.

TUCAN/CARDINAL and DRAL participate in a common pathway for modulation of NF-κB activation

Proteins containing the caspase recruiting domain (CARD) have emerged as critical regulators of different signal transduction pathways, including those controlling apoptosis and activation of necrosis factor (NF)‐κB transcription factor. TUCAN/CARDINAL is a recently identified CARD‐containing protein involved in regulation of caspases and NF‐κB activation. We find that TUCAN/CARDINAL associates with DRAL, a p53‐responsive gene implicated in induction of apoptosis. We also show that, whereas TUCAN/CARDINAL exerts a suppressive effect on NF‐κB activity, expression of DRAL results in enhancement of NF‐κB activation. Thus, our observations suggest that DRAL and TUCAN/CARDINAL may participate in a regulatory mechanism that coordinates cellular responses controlled by NF‐κB transcription factor.

Alternative splicing of CARMA2/CARD14 transcripts generates protein variants with differential effect on NF-κB activation and endoplasmic reticulum stress-induced cell death.

The caspase recruitment domain (CARD)‐containing proteins CARMA1‐3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF‐κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF‐κB in a TRAF2‐dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF‐κB activation and apoptosis. J. Cell. Physiol. 226: 3121–3131, 2011.

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity.

A20 is a negative regulator of BCL10- and CARMA3-mediated activation of NF-κB

The molecular complex containing CARMA proteins, BCL10 and TRAF6 has been identified recently as a key component in the signal transduction pathways that regulate activation of the nuclear factor κB (NF-κB) transcription factor. Here, we report that the inducible protein A20 negatively regulates these signaling cascades by means of its deubiquitylation activity. We show that A20 perturbs assembly of the complex containing CARMA3, BCL10 and IKKγ/NEMO, thereby suppressing activation of NF-κB. Together, our results further define the molecular mechanisms that control activation of NF-κB and reveal a function for A20 in the regulation of CARMA and BCL10 activity in lymphoid and non-lymphoid cells.

The seventh ring: Exploring TRAF7 functions

Tumor necrosis factor receptor‐associated factors (TRAFs) have been discovered and characterized by their capacity to link tumor necrosis factor receptors (TNFR) family proteins to signaling pathways that transduce the cellular effects mediated by TNF family ligands. There are seven known mammalian TRAF proteins (TRAF1–7), that share a domain organization made of a modular structure, characteristic of adaptor proteins whose function is to link structurally dissimilar factors. Functionally, TRAF proteins mediate the assembly of cytoplasmic signal transducers and regulatory molecules downstream of receptors complexes. Despite the similarities in the signaling pathways activated by the different TRAF proteins, each appears to play distinct physiological roles. TRAF7 is the last member of the TRAF family that has been identified. Yet, the functional characterization of TRAF7 presents some aspects still obscure and poorly defined, making this protein arguably the most mysterious member of the family. In fact, recent data indicate that TRAF7 is involved in signal transduction pathways that lead either to activation or repression of NF‐κB transcription factor. In addition, TRAF7 regulates activation of cellular stress pathways, as well as unconventional ubiquitination events and differentiation of muscle tissue. In this review, we try to summarize the most recent advances in our understanding of TRAF7 function and the biological processes of this protein is involved in. J. Cell. Physiol. 227: 1280–1284, 2012.

TRAF7 promotes K29-linked polyubiquitination of IKKγ/nemo and p65/RELA and represses NF-kB activation

Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity.