Dataset Information

Proteomics of testis from SPPL2c KO mice

ABSTRACT: Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we have analysed the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We identified proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localization, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles and are organised in different high molecular-weight complexes. Unexpectedly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c-deficiency leads to a loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. Additionally, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identified the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease of intracellular Ca2+ levels in elongated spermatids that likely contributes to the compromised male germ cell differentiation and function of SPPL2c-/- mice.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Testis

SUBMITTER: Stephan Mueller

LAB HEAD: Bernd Schröder

PROVIDER: PXD011922 | Pride | 2019-02-12

REPOSITORIES: Pride

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Publications

The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban.

Niemeyer Johannes J Mentrup Torben T Heidasch Ronny R Müller Stephan A SA Biswas Uddipta U Meyer Rieke R Papadopoulou Alkmini A AA Dederer Verena V Haug-Kröper Martina M Adamski Vivian V Lüllmann-Rauch Renate R Bergmann Martin M Mayerhofer Artur A Saftig Paul P Wennemuth Gunther G Jessberger Rolf R Fluhrer Regina R Lichtenthaler Stefan F SF Lemberg Marius K MK Schröder Bernd B

EMBO reports 20190207 3

Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though part ...[more]

PMID: 30733280

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Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

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