Yuko S. Askew

An Intracellular Serpin Regulates Necrosis by Inhibiting the Induction and Sequelae of Lysosomal Injury

Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.

SERPINB11 Is a New Noninhibitory Intracellular Serpin COMMON SINGLE NUCLEOTIDE POLYMORPHISMS IN THE SCAFFOLD IMPAIR CONFORMATIONAL CHANGE

SERPINB11, the last of 13 human clade B serpins to be described, gave rise to seven different isoforms. One cDNA contained a premature termination codon, two contained splice variants, and four contained full-length open reading frames punctuated by eight single nucleotide polymorphisms (SNPs). The SNPs encoded amino acid variants located within the serpin scaffold but not the reactive site loop (RSL). Although the mouse orthologue, Serpinb11, could inhibit trypsin-like peptidases, SERPINB11 showed no inhibitory activity. To determine whether the human RSL targeted a different class of peptidases or the serpin scaffold was unable to support inhibitory activity, we synthesized chimeric human and mouse proteins, in which the RSLs had been swapped. The human RSL served as a trypsin inhibitor when supported by mouse scaffold sequences. Conversely, the mouse RSL on the human scaffold showed no inhibitory activity. These findings suggested that variant residues in the SERPINB11 scaffold impaired serpin function. SDS-PAGE analysis supported this notion as RSL-cleaved SERPINB11 was unable to undergo the stressed-to-relaxed transition typical of inhibitory type serpins. Mutagenesis studies supported this hypothesis, since the reversion of amino acid sequences in helices D and I to those conserved in other clade B serpins partially restored the ability of SERPINB11 to form covalent complexes with trypsin. Taken together, these findings suggested that SERPINB11 SNPs encoded amino acids in the scaffold that impaired RSL mobility, and HapMap data showed that the majority of genomes in different human populations harbored these noninhibitory SERPINB11 alleles. Like several other serpin superfamily members, SERPINB11 has lost inhibitory activity and may have evolved a noninhibitory function.

Selective conservation of the RSL-encoding, proteinase inhibitory-type, clade L serpins in Caenorhabditis species

Serpins are a highly conserved superfamily of serine and papain-like cysteine proteinase inhibitors that are divided phylogenetically into clades. Serpins also can be divided anatomically into those that reside predominately outside or inside cells. While the activities of the extracellular serpins are well understood, the biological functions, as well as the overall distribution of the intracellular (serpinIC) serpins is less well defined. Conceivably, the biological function of the serpinsIC might be revealed by analysis of species with genomes of lower complexity. To this end, we sought to define the clade L serpin repertoire of Caenorhabditis elegans and other nematode species. Analysis of the C. elegans genome revealed the presence of 9 serpin genes. Five genes encoded for full-length serpins with functional reactive site loops (RSL). By definition, these genes were designated proteinase inhibitory-type, RSL-encoding serpins. Four of the C. elegans genes encoded for proteins without an RSL or transcripts with premature termination codons. The high percentage of non-RSL encoding to RSL-encoding serpin genes suggested that the former served a unique biological function rather than residing in the genome as simple pseudogenes. If this hypothesis was correct, we expected these non-RSL encoding genes to be conserved precisely in other Caenorhabditis species. However, in contrast to the RSL-encoding serpins that were well conserved and segregated into 3 sub-clades, we failed to detect non-RSL encoding serpin orthologues in the genomes of Caenorhabditis briggsae and Caenorhabditis remanei. These data suggested that unlike their RSL-encoding paralogues, the relatively high percentage of non-RSL encoding serpins in C. elegans was a vestige of recent duplication events and these latter genes were unlikely to serve essential functions in Caenorhabditis species.