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2.
EMBO Rep ; 20(3)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30733280

RESUMO

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 partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c -/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c -/- mice.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/enzimologia , Células Germinativas/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Células HeLa , Homeostase , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Espermátides/metabolismo , Especificidade por Substrato , Testículo/enzimologia
3.
EMBO Rep ; 20(3)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30733281

RESUMO

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas SNARE/metabolismo , Acrossomo/metabolismo , Animais , Biocatálise , Regulação para Baixo , Glicômica , Glicoproteínas/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transporte Proteico , Proteólise , Espermátides/metabolismo , Frações Subcelulares/metabolismo , Especificidade por Substrato
4.
EMBO Rep ; 20(3): e46451, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-32048434

RESUMO

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.


Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Proteases/genética , Proteínas SNARE/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento , Acrossomo/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Ácido Aspártico Endopeptidases/metabolismo , Citocinese/genética , Retículo Endoplasmático/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosilação , Complexo de Golgi/genética , Hepatite/genética , Hepatite/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Proteômica , Especificidade por Substrato , Testículo/metabolismo , Proteínas de Transporte Vesicular/genética
5.
Exp Cell Res ; 357(1): 40-50, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28442266

RESUMO

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Linhagem Celular , Humanos , Lisossomos/metabolismo , Transporte Proteico , Solubilidade , Rede trans-Golgi/metabolismo
6.
J Biol Chem ; 291(1): 318-33, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26574544

RESUMO

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of ß-secretase (ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III ß-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.


Assuntos
Membrana Celular/enzimologia , Neuregulina-1/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Peptídeo C/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação/genética , Neurônios/metabolismo , Peptídeos/química , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína , Ratos , Esquizofrenia/genética , Especificidade por Substrato
7.
Mol Cell Proteomics ; 14(6): 1584-98, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25827571

RESUMO

Signal peptide peptidase-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Complexo de Golgi/metabolismo , Glicosilação , Células HEK293 , Humanos
8.
EMBO J ; 33(24): 2890-905, 2014 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-25354954

RESUMO

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Regulação da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional
9.
J Biol Chem ; 287(52): 43401-9, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23132852

RESUMO

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Produtos do Gene env/metabolismo , Sinais Direcionadores de Proteínas , Proteólise , Vírus Espumoso dos Símios/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Produtos do Gene env/genética , Células HEK293 , Humanos , Vírus Espumoso dos Símios/genética
10.
J Biol Chem ; 287(7): 5156-63, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22194595

RESUMO

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Dicroísmo Circular/métodos , Células HEK293 , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Mutagênese , Estrutura Terciária de Proteína
11.
J Biol Chem ; 283(44): 30121-8, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18768471

RESUMO

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Ácido Aspártico Endopeptidases/genética , Mutação , Motivos de Aminoácidos , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/química , Catálise , Linhagem Celular , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/química , Estrutura Terciária de Proteína , Temperatura , Fator de Necrose Tumoral alfa/metabolismo
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