BACKGROUND: PSAP encodes saposin C, the co-activator of glucocerebrosidase, encoded by GBA. GBA mutations are associated with idiopathic/isolated REM sleep behavior disorder (iRBD), a prodromal stage of synucleinopathy. OBJECTIVE: To examine the role of PSAP mutations in iRBD. METHODS: We fully sequenced PSAP and performed Optimized Sequence Kernel Association Test in 1,113 iRBD patients and 2,324 controls. We identified loss-of-function (LoF) mutations, which are very rare in PSAP, in three iRBD patients and none in controls (uncorrected p = 0.018). RESULTS: Two variants were stop mutations, p.Gln260Ter and p.Glu166Ter, and one was an in-frame deletion, p.332_333del. All three mutations have a deleterious effect on saposin C, based on in silico analysis. In addition, the two carriers of p.Glu166Ter and p.332_333del mutations also carried a GBA variant, p.Arg349Ter and p.Glu326Lys, respectively. The co-occurrence of these extremely rare PSAP LoF mutations in two (0.2%) GBA variant carriers in the iRBD cohort, is unlikely to occur by chance (estimated co-occurrence in the general population based on gnomAD data is 0.00035%). Although none of the three iRBD patients with PSAP LoF mutations have phenoconverted to an overt synucleinopathy at their last follow-up, all manifested initial signs suggestive of motor dysfunction, two were diagnosed with mild cognitive impairment and all showed prodromal clinical markers other than RBD. Their probability of prodromal PD, according to the Movement Disorder Society research criteria, was 98% or more. CONCLUSION: These results suggest a possible role of PSAP variants in iRBD and potential genetic interaction with GBA, which requires additional studies.

• Klíčová slova
• GBA, PSAP, Parkinson’s disease, REM sleep behavior disorder, genetics, glucocerebrosidase, saposin C,
• MeSH
• glukosylceramidasa genetika   MeSH
• lidé MeSH
• Parkinsonova nemoc * komplikace   MeSH
• porucha chování v REM spánku * diagnóza   MeSH
• saposiny genetika   MeSH
• synukleinopatie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosylceramidasa MeSH
• PSAP protein, human MeSH Prohlížeč
• saposiny MeSH

OBJECTIVE: To examine the role of genes identified through genome-wide association studies (GWASs) of Parkinson disease (PD) in the risk of isolated REM sleep behavior disorder (iRBD). METHODS: We fully sequenced 25 genes previously identified in GWASs of PD in a total of 1,039 patients with iRBD and 1,852 controls. The role of rare heterozygous variants in these genes was examined with burden tests. The contribution of biallelic variants was further tested. To examine the potential effect of rare nonsynonymous BST1 variants on the protein structure, we performed in silico structural analysis. Finally, we examined the association of common variants using logistic regression adjusted for age and sex. RESULTS: We found an association between rare heterozygous nonsynonymous variants in BST1 and iRBD (p = 0.0003 at coverage >50× and 0.0004 at >30×), driven mainly by 3 nonsynonymous variants (p.V85M, p.I101V, and p.V272M) found in 22 (1.2%) controls vs 2 (0.2%) patients. All 3 variants seem to be loss-of-function variants with a potential effect on the protein structure and stability. Rare noncoding heterozygous variants in LAMP3 were also associated with iRBD (p = 0.0006 at >30×). We found no association between rare heterozygous variants in the rest of genes and iRBD. Several carriers of biallelic variants were identified, yet there was no overrepresentation in iRBD. CONCLUSION: Our results suggest that rare coding variants in BST1 and rare noncoding variants in LAMP3 are associated with iRBD. Additional studies are required to replicate these results and to examine whether loss of function of BST1 could be a therapeutic target.

• MeSH
• ADP-ribosylcyklasa genetika   MeSH
• CD antigeny genetika   MeSH
• celogenomová asociační studie MeSH
• databáze genetické MeSH
• genetická variace MeSH
• GPI-vázané proteiny genetika   MeSH
• heterozygot MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové glykoproteiny asociované s lyzozomy genetika   MeSH
• nádorové proteiny genetika   MeSH
• počítačová simulace MeSH
• polysomnografie MeSH
• porucha chování v REM spánku epidemiologie   genetika   MeSH
• sekundární struktura proteinů MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADP-ribosyl cyclase 2 MeSH Prohlížeč
• ADP-ribosylcyklasa MeSH
• CD antigeny MeSH
• GPI-vázané proteiny MeSH
• LAMP3 protein, human MeSH Prohlížeč
• membránové glykoproteiny asociované s lyzozomy MeSH
• nádorové proteiny MeSH

Genome integrity and cell survival are dependent on proper replication stress response. Multiple repair pathways addressing obstacles generated by replication stress arose during evolution, and a detailed understanding of these processes is crucial for treatment of numerous human diseases. Here, we investigated the strong negative genetic interaction between two proteases involved in the DNA replication stress response, yeast Wss1 and Ddi1. While Wss1 proteolytically acts on DNA-protein crosslinks, mammalian DDI1 and DDI2 proteins remove RTF2 from stalled forks via a proposed proteasome shuttle hypothesis. We show that the double-deleted Δddi1, Δwss1 yeast strain is hypersensitive to the replication drug hydroxyurea and that this phenotype can be complemented only by catalytically competent Ddi1 protease. Furthermore, our data show the key involvement of the helical domain preceding the Ddi1 protease domain in response to replication stress caused by hydroxyurea, offering the first suggestion of this domain's biological function. Overall, our study provides a basis for a novel dual protease-based mechanism enabling yeast cells to counteract DNA replication stress.

• Klíčová slova
• DNA replication stress, Ddi1, Hydroxyurea, Protease, Wss1, Yeast,
• MeSH
• DNA metabolismus   MeSH
• hydroxymočovina toxicita   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• replikace DNA * MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   enzymologie   genetika   metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDI1 protein, S cerevisiae MeSH Prohlížeč
• DNA MeSH
• hydroxymočovina MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• WSS1 protein, S cerevisiae MeSH Prohlížeč

The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains.

• MeSH
• interakční proteinové domény a motivy * MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multigenová rodina MeSH
• poškození DNA * MeSH
• proteomika metody   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• ubikvitin metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDI1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• ubikvitin MeSH

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

• MeSH
• aminokyselinové motivy MeSH
• aspartátové proteasy chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mapování interakce mezi proteiny MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• polyubikvitin metabolismus   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• strukturní homologie proteinů * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové proteasy MeSH
• DDI2 protein, human MeSH Prohlížeč
• polyubikvitin MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny MeSH