Chul-Woong Chung

Cleavage of Bax is mediated by caspase‐dependent or ‐independent calpain activation in dopaminergic neuronal cells: protective role of Bcl‐2

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress‐specific protease activation pathway exists, and to what extent Bcl‐2 plays a role in preventing drug‐induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase‐dependent apoptosis while a dopaminergic neurotoxin, MPP+ largely induced caspase‐independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS‐ and MPP+‐induced cell death, Bax was cleaved into an 18‐kDa fragment. This 18‐kDa fragment appeared only in the mitochondria‐enriched heavy membrane fraction of STS‐treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP+‐treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [35S]methionine‐labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP+‐ and STS‐induced Bax cleavage. Intriguingly, overexpression of baculovirus‐derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS‐ but not MPP+‐induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP+‐induced but not from STS‐induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl‐2 prevented both STS‐ and MPP+‐induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl‐2 may play a protective role by primarily blocking drug‐induced caspase or calpain activity in dopaminergic neuronal cells.

Pro-apoptotic function of calsenilin/DREAM/KChIP3

Apoptotic cell death and increased production of amyloid β peptide (Aβ) are pathological features of Alzheimer’s disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro‐apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide‐induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+‐ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3‐induced apoptosis was suppressed by caspase inhibitor z‐VAD and by Bcl‐xL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPsw) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C‐terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Aβ42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis‐associated Aβ42 production of calsenilin/DREAM/KChIP3.

E2-25K/Hip-2 regulates caspase-12 in ER stress–mediated Aβ neurotoxicity

Amyloid-β (Aβ) neurotoxicity is believed to contribute to the pathogenesis of Alzheimers disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Aβ neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)–resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Aβ increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2–mediated cell death. Finally, we find that E2-25K/Hip-2–deficient cortical neurons are resistant to Aβ toxicity and to the induction of ER stress and caspase-12 expression by Aβ. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress–mediated Aβ neurotoxicity.

Role of FLASH in caspase-8-mediated activation of NF-κB: Dominant-negative function of FLASH mutant in NF-κB signaling pathway

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-α-induced activation of NF-κB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-α-induced activation of NF-κB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-α for NF-κB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-κB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-κB activation triggered by TNF-α and caspase-8. In addition, expression of an NF-κB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-α and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-κB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-α-induced activation of NF-κB via FLASH.

Inactivation of farnesyltransferase and geranylgeranyltransferase I by caspase-3: Cleavage of the common α subunit during apoptosis

Caspase plays an important role in apoptosis. We report here that farnesyltransferase/geranylgeranyltransferase (FTase/GGTase)-α, a common subunit of FTase (α/βFTase) and GGTase I (α/βGGTase), was cleaved by caspase-3 during apoptosis. FTase/GGTase-α (49 kDa) was cleaved to 35 kDa (p35) in the Rat-2/H-ras, W4 and Rat-1 cells treated with FTase inhibitor (LB42708), anti-Fas antibody and etoposide, respectively. This cleavage was inhibited by caspase-inhibitors (YVAD-cmk, DEVD-cho). Serial N-terminal deletions and site-directed mutagenesis showed that Asp59 of FTase/GGTase-α was cleaved by caspase-3. The common FTase/GGTase-α subunit, but not the β subunits, of the FTase or GGTase I protein complexes purified from baculovirus-infected SF-9 cells was cleaved to be inactivated by purified caspase-3. In contrast, FTase mutant protein complex [(D59A)α/βFTase] was resistant to caspase-3. Expression of either the cleavage product (60-379) or anti-sense of FTase/GGTase-α induced cell death in Rat-2/H-ras cells. Furthermore, expression of (D59A)FTase/GGTase-α mutant significantly desensitized cells to etoposide-induced death. Taken together, we suggest that cleavage of prenyltransferase by caspase contributes to the progression of apoptosis.

Atypical role of proximal caspase-8 in truncated Tau-induced neurite regression and neuronal cell death.

Abnormal Tau protein is known to be closely associated with several neurodegenerative diseases. Previously, we showed that Tau was cleaved by caspase-3 to generate the cleavage product lacking the C-terminus (DeltaTau-1) during neuronal cell death. Here we characterized caspase-8-dependent neurotoxicity of the truncated Tau. Introduction of DeltaTau-1 into primary hippocampal neurons induced loss of neurites in a caspase-dependent manner. Caspase-8 and -6 were proteolytically activated during DeltaTau-1-triggered neuronal cell death, which was suppressed by IETD-fmk, caspase-8 inhibitor. Direct targeting of caspase-8 and its associated FADD with antisense approaches and transient expression of their dominant-negative mutants reduced DeltaTau-1-induced apopotosis. Cells deficient in caspase-8, but not caspase-3, became sensitized to DeltaTau-1-mediated toxicity upon reconstitution with caspase-8. In addition, ectopic expression of mitochondrial antiapoptotic Bcl-2, Bcl-X(L), or inactive caspase-9 short form suppressed DeltaTau-1 toxicity. These results suggest that the truncated Tau protein activates proximal caspase-8 through FADD as a necessary step leading to neuronal cell death and neurite regression, contributing to the progression of abnormal Tau-associated neurodegeneracy.

Considering cell‐based assays and factors for genome‐wide high‐content functional screening

Abstract Recently, great advance is achieved in the field of genome‐wide functional screening using cell‐based assay. Here, we briefly introduce well‐established and typical cell‐based assays of GPCR and some parameters which should be considered for genome‐wide functional screening. Because of characters and importance of GPCR as drug targets, several ways of assay systems were devised. Among them, high‐content screening (HCS) that is based on the analysis of image by confocal microscope is becoming favorite choice. The advances in this technology have been driven exclusively by industry for their convenience. Now, it is turn for academy to define more detail signaling networks via HCS using cDNA or siRNA libraries at genome‐wide level. By isolating novel signaling mediators using cDNA or siRNA library, and postulating them as new candidates for therapeutic target, more understanding about life science and more increased chances to develop therapeutics against human disease will be achieved.