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The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER in Caenorhabditis Elegans

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The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER in Caenorhabditis Elegans GENETICS | INVESTIGATION The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER in Caenorhabditis elegans Richard M. Silverman, Erin E. Cummings, Linda P. O’Reilly, Mark T. Miedel, Gary A. Silverman, Cliff J. Luke, David H. Perlmutter, and Stephen C. Pak1 Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh of University of Pittsburgh Medical Center and Magee–Womens Hospital Research Institute, Pittsburgh, Pennsylvania 15224 ABSTRACT Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)–Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER. In a previous study, nematodes expressing a homologous mutation in the endogenous Caenorhabditis elegans serpin, srp-2,werereportedtomodeltheERproteotoxicityinducedbyanallele of mutant neuroserpin. Our results suggest that SRP-2 lacksaclassicalN-terminalsignalpeptideandisamemberofthe intracellular serpin family. Using confocal imaging and an ER colocalization marker, we confirmed that GFP-tagged wild-type SRP-2 localized to the cytosol and not the ER. Similarly, the aggregation- prone SRP-2 mutant formed intracellular inclusions that localized to the cytosol. Interestingly, wild-type SRP-2,targetedtotheERbyfusion to a cleavable N-terminal signal peptide, failedtobesecretedandaccumulatedwithintheERlumen.ThisERretentionphenotypeistypical of other obligate intracellular serpins forced to translocate across the ER membrane. Neuroserpin is a secreted protein that inhibits trypsin- like proteinase. SRP-2 is a cytosolic serpin that inhibits lysosomal cysteine peptidases. We concluded that SRP-2 is neither an ortholog nor a functional homolog of neuroserpin. Furthermore, animals expressing an aggregation-prone mutation in SRP-2 do not model the ER proteotoxicity associated with FENIB. KEYWORDS ER stress; familial encephalopathy with neuroserpin inclusions; SRP-2; neuroserpin; proteostasis EMBERS of the serine proteinase inhibitor (serpin) Due to the metastability of the native serpin fold, which is Msuperfamily are best known as physiological regula- crucial for its inhibitory activity, even single nonsynonymous tors of proteolytic cascades associated with coagulation, amino acid changes make these proteins highly susceptible to thrombolysis, inflammation, and cell death (Silverman misfolding and aggregation (Huntington 2006). This aggregation- et al. 2010; Whisstock et al. 2010). To accomplish this task prone phenotype is most evident in patients with a1-antitrypsin serpins fold into a highly conserved metastable structure (a1AT)/SERPINA1 deficiency (ATD). The most common mu- consisting of three b-sheets, eight to nine a-helices, and tation, Z (E342K), impairs the latter stages of serpin folding an exposed reactive center loop (RCL), which serves as bait and facilitates domain swaps between monomers, yielding for target proteinases (Huber and Carrell 1989; Irving et al. oligomers and higher-order polymers (Yamasaki et al. 2011). 2000). After a target proteinase binds and cleaves its cog- Although a small percentage of the monomers of the mutant nate RCL, strain on the serpin scaffold is relieved, thereby protein (ATZ) are constitutively secreted via the endoplasmic triggering a conformational change that traps the serpin and reticulum (ER)–Golgi pathway, toxic monomers and higher- proteinase in a covalent complex (Huntington et al. 2000). order species accumulate within the ER of hepatocytes (Perlmutter 2002, 2011; Silverman et al. 2013). Ultimately, these ER-retained Copyright © 2015 by the Genetics Society of America ATZ species, which appear as periodic-acid Schiff positive doi: 10.1534/genetics.115.176180 (PAS+), diastase-resistant inclusions in histological liver Manuscript received January 23, 2015; accepted for publication March 17, 2015; published Early Online March 18, 2015. specimens, lead to cirrhosis and in some cases hepatocellular Supporting information is available online at http://www.genetics.org/lookup/suppl/ carcinoma (Perlmutter 2006, 2007; Silverman et al. 2013). doi:10.1534/genetics.115.176180/-/DC1. 1Corresponding author: Children’s Hospital of Pittsburgh of University of Pittsburgh Similar types of mutations have resulted in the intracel- Medical Center, 4401 Penn Ave., Pittsburgh, PA 15224. E-mail: [email protected] lular accumulation of other extracellular serpins such as Genetics, Vol. 200, 207–219 May 2015 207 antithrombin (SERPINC1), C1 esterase inhibitor (SERPING1), being transcribed pseudogenes or noninhibitory variants a1-antichymotrypsin (SERPINA3), and neuroserpin (NS/ (Pak et al. 2004, 2006; Luke et al. 2006, 2007). Similar SERPINI1) (Roussel et al. 2011). Collectively, these disorders, to the clade B serpins, these nematode serpins lacked a sig- which all involve proteins secreted via the classical ER–Golgi nal peptide and the N- and C-terminal extensions typical of pathway, have been designated serpinopathies and are char- serpins transiting the conventional ER–Golgi secretory acterized by (1) a loss-of-function phenotype due to decreased pathway. Green fluorescent protein (GFP)–nematode ser- circulating levels of the inhibitor and (2) a gain-of-function pin fusions show a diffuse cytoplasmic appearance and no phenotype due to cellular proteotoxicity associated with re- evidence of secretion into the intestinal cell lumen, the tention of misfolded proteins within the ER of the synthesizing pseudocoelomic space, or the cuticle (Pak et al. 2004, cells (Carrell and Lomas 1997, 2002; Carrell 2005). 2006; Luke et al. 2006, 2007). In 2001, the serpin nomenclature committee divided the Recently, one of the C. elegans serpin genes, srp-2, was 36 human serpins into nine clades (A–I) (Silverman et al. cloned, mutagenized, and reintroduced in the nematode 2001). Unique among this group are the clade B serpins, germline by biolistic transformation (Schipanski et al. which belong to the larger evolutionarily well-conserved 2013). This mutation was introduced into a conserved res- group of intracellular serpins (Remold-O’Donnell 1993; idue within the serpin scaffold and is homologous to a highly Silverman et al. 2004). In comparison to the other human polymerogenic mutation found in some patients with the ser- serpins (clades A and C–I), which are all secreted proteins pinopathy, familial encephalopathy with neuroserpin inclusion containing cleavable N-terminal signal peptides, the intra- bodies (FENIB) (Davis et al. 2002; Gooptu and Lomas 2009). cellular serpins (clade B) lack a recognizable secretion signal The authors conclude that this transgenic strain accumulates as well as N- and C-terminal extensions. Generally, these mutant SRP-2 in the ER and therefore serves as an inverte- serpins possess a nucleo-cytosolic subcellular distribution brate model of FENIB. Moreover, other investigators have used (Bird et al. 2001; Silverman et al. 2004). However, one these transgenic animals to study the relationship between member of the intracellular serpin family, chicken ovalbumin, a luminal misfolded ER protein and the unfolded protein re- is secreted by the chicken oviduct (Palmiter et al. 1978). Also, sponse (UPR) and ER-associated degradation (ERAD) path- but to a much lesser extent and only under certain conditions, ways under different experimental conditions (Denzel et al. plasminogen-activator type 2 (PAI2/SERPINB2) appears to be 2014; Hou et al. 2014). However, using colocalization studies inefficiently secreted (Belin 1993; Belin et al. 2004). Neither and comparisons to the canonical serpinopathy caused by ovalbumin nor PAI2/SERPINB2 possesses a cleavable N- ATZ, we show unequivocally that wild-type and mutant terminal signal peptide (Palmiter et al. 1978). Mutagenesis SRP-2 reside in the cytosol and not in the ER. While these studies suggest that embedded hydrophobic motifs in the animals expressing mutant SRP-2 could be used to study the N terminus facilitate secretion with that of PAI2/SERPINB2 effects of cytosolic serpin polymerization, the markedly dif- being very inefficient (Tabe et al. 1984; Belin et al. 2004). ferent expression patterns, inhibitory profiles, and subcellu- Although some of these secreted intracellular serpins pos- lar localization of SRP-2 compared to those of human NS/ sess complex N-linked carbohydrates suggestive of Golgi SERPINI1 confound the ability to use these animals to model processing, secretion still occurs in the presence of tunicamycin accurately the interaction between aging and ER overload in (Keller and Swank 1978) and may involve unconventional patients with the serpinopathy, FENIB. Furthermore, inter- signal pathway(s) similar to those used by FGF-2 and IL-1b esting experimental results regarding the effects of mutant (Nickel and Rabouille 2009; Giuliani et al. 2011; Malhotra SRP-2 on ER proteostasis pathways should be viewed from 2013).Indeed,forcedexpressionofwild-typeproteaseinhibi- the perspective that this proteotoxic stress originated from tor 6 (PI6)/SERPINB6 and MASPIN/SERPINB5 into the ER– within the cytosol and not the ER (Denzel et al. 2014; Hou Golgi pathway by fusing an N-terminal signal peptide leads to et al. 2014). overt polymerization and ER retention (Scott et al. 1996; Teoh et al. 2010). None of the
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