Lisa M. Ellerby

Pro-caspase-3 Is a Major Physiologic Target of Caspase-8

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (k cat/K m ) of 8.7 × 105 and 2.8 × 105 m −1 s−1, respectively. These rates are of sufficient magnitude to indicate direct processingin vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

Caspase Cleavage of Gene Products Associated with Triplet Expansion Disorders Generates Truncated Fragments Containing the Polyglutamine Tract

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

A second cytotoxic proteolytic peptide derived from amyloid β-proteinprecursor

The amyloid β-protein precursor gives rise to the amyloid β-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid β-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid β-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid β-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.

Abstract : X‐linked spinal and bulbar muscular atrophy (SBMA), Kennedys disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease‐associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity ; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase‐3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenrative diseases associated with polyglutamine repeat expansions.

Cleavage of Atrophin-1 at Caspase Site Aspartic Acid 109 Modulates Cytotoxicity

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

Determinants of Cytochrome c Pro-apoptotic Activity THE ROLE OF LYSINE 72 TRIMETHYLATION

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72,N-ε-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome cexpressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

Biomolecular Materials Based on Sol-Gel Encapsulated Proteins

The proteins copper-zinc superoxide dismutase (CuZnSOD), cytochrome c, myoglobin, hemoglobin, and bacterio-rhodopsin are encapsulated in stable, optically transparent, porous, silica glass matrices prepared by the sol-gel method such that the biomolecules retain their characteristic reactivities and spectroscopic properties. The resulting glasses allow transport of small molecules into and out of the glasses at reasonable rates but retain the protein molecules within their pores. The transparency of the glasses enables the chemical reactions of the immobilized proteins to be monitored by means of changes in their visible absorption spectra. Silica glasses containing the immobilized proteins have similar reactivities and spectroscopic properties to those found for the proteins in solution. The enzymes glucose oxidase and peroxidase were also encapsulated in transparent silica glass matrices. Upon exposure to glucose solutions, a colored glass is formed that can be used as the active element in a solid state optically based glucose sensor.

Encapsulation and reactivity of the enzyme oxalate oxidase in a sol-gel derived glass: Code: GP4

The enzymes oxalate oxidase and peroxidase are encapsulated in stable, optically transparent, porous silica glass matrices synthesized under mild conditions using novel sol-gel synthetic techniques. The large enzymes are immobilized, but smaller molecules such as oxalate ions pass readily through the porous glass. Upon exposure to oxalate solutions, a colored glass is formed whose absorption spectrum and changes of absorbance with time are measured. The sensitivity of the response and the time-dependence of the response are discussed.