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Schematic overview of subcellular compartments and respective paradigmatic proteins that showed significant regulation (≥1.6-fold, p value ≤0.05) upon SIL1 depletion in HEK293 cells. The gene name color coding indicates increased (blue) and decreased (red) protein expression, as compared to non-depleted HEK293 control cells. Corresponding ratios were determined by label-free quantitative mass spectrometry as described in detail in the supplemental methods
Source publication
SIL1 acts as nucleotide exchange factor for the endoplasmic reticulum chaperone BiP. Mutations of SIL1 cause Marinesco-Sjögren syndrome (MSS), a neurodegenerative disorder. Moreover, a particular function of SIL1 for etiopathology of amyotrophic lateral sclerosis (ALS) was highlighted, thus declaring the functional SIL1-BiP complex as a modifier fo...
Citations
... The human spectral library is queried with 18 search files from the human HEK293 cell line [Roos et al. (2016); PRIDE ID: PXD001197] aligning our study with the spectral benchmarking by Wang et al. (2020) that already compared msSLASH and SpectraST. ...
Motivation:
Deep learning has moved to the forefront of tandem mass spectrometry-driven proteomics and authentic prediction for peptide fragmentation is more feasible than ever. Still, at this point spectral prediction is mainly used to validate database search results or for confined search spaces. Fully predicted spectral libraries have not yet been efficiently adapted to large search space problems that often occur in metaproteomics or proteogenomics.
Results:
In this study, we showcase a workflow that uses Prosit for spectral library predictions on two common metaproteomes and implement an indexing and search algorithm, Mistle, to efficiently identify experimental mass spectra within the library. Hence, the workflow emulates a classic protein sequence database search with protein digestion but builds a searchable index from spectral predictions as an in-between step. We compare Mistle to popular search engines, both on a spectral and database search level, and provide evidence that this approach is more accurate than a database search using MSFragger. Mistle outperforms other spectral library search engines in terms of run time and proves to be extremely memory efficient with a 4 to 22-fold decrease in RAM usage. This makes Mistle universally applicable to large search spaces, e.g. covering comprehensive sequence databases of diverse microbiomes.
Availability:
Mistle is freely available on GitHub at https://github.com/BAMeScience/Mistle.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... Elevated secretion of this protein identified in PORCN-patient derived fibroblasts might correlate (i) with oxidative stress as indicated by concomitant increased secretion of Peroxiredoxin-6 and (ii) with cytoskeletal changes which are indicated by the results of our biochemical studies. Cytoskeletal changes were already associated with other multisystemic diseases originating from altered ER-related protein-processing capacity [43] and have an impact in the ethology of different disorders including neurological diseases [30,44]. As for impaired protein processing avenues, also based on the known interaction of cytoskeletal proteins with small molecules [45], drugs targeting the cytoskeleton are currently being tested showing promising effects [46]. ...
Background
Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.
Results
We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.
Conclusions
Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.
... IPA identified 170 significantly affected "Canonical Pathways" (Tables 5 and S6). This large number of disrupted pathways is in line with previous studies, which showed that proteins with altered expression belong to all cellular compartments [23]. Among the affected canonical pathways, IPA confirmed phagosome, beta oxidation of fatty acids, TCA cycle, and degradation of amino acids in accordance with KEGG enrichment analysis (Table 4). ...
Marinesco–Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.
... Notably, pathogenic missense variants of Sil1 lead to a disruption of the SIL1-POC1A interaction which is in turn associated with centrosome disintegration [289]. Further morphological and biochemical studies on an in vitro model (Sil1-depleted HEK293 cells) utilizing electron microscopy and unbiased proteomic profiling revealed structural changes of the ER including the nuclear envelope and mitochondrial degeneration that closely mimic pathological alterations in MSS as well as indicated that proteins involved in cytoskeletal organization, vesicular transport, mitochondrial function, and neurological processes contribute to Sil1 pathophysiology [285]. Moreover, a particular function of Sil1 for etiopathology of two neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and Alzheimer disease was highlighted, thus declaring the functional Sil1-BiP complex as a modifier for neurodegenerative disorders [282,283,287]. ...
The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in protein biogenesis, calcium (Ca2+) homeostasis, and signal transduction. Among the roughly one hundred components, which are involved in protein import and protein folding or assembly, two components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides a conduit for Ca2+ ions from the ER lumen to the cytosol. The second component, the Hsp70-type molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles in protein folding and assembly (hence its name), protein import, cellular Ca2+ homeostasis, and various intracellular signal transduction pathways. For the purpose of this review, we focus on these two components, their relevant allosteric effectors and on the question of how their respective functional cycles are linked in order to reconcile the apparently contradictory features of the ER membrane, selective permeability for precursor polypeptides, and impermeability for Ca2+. The key issues are that the Sec61 complex exists in two conformations: An open and a closed state that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both directions in cooperation with different co-chaperones. While the open Sec61 complex forms an aqueous polypeptide-conducting- and transiently Ca2+-permeable channel, the closed complex is impermeable even to Ca2+. Therefore, we discuss the human hereditary and tumor diseases that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective polypeptide-impermeability and/or aberrant Ca2+-permeability.
... Like MSS patients, woozy and Sil1 Gt mice develop severe myopathy and muscle atrophy [28,35]. Early signs of muscle disease, including non-subsarcolemmal nuclei and autophagic vacuoles (Table 2), can be seen in woozy mice already at 16 weeks of age, while severe myopathy develops later [28,33,36]. Muscle cells also present alterations of the Golgi structure, swollen mitochondria, proliferated sarcoplasmic reticulum and chromatin condensation. ...
... Cell lines with reduced SIL1 expression develop cell pathological and molecular features consistent with the physiopathology of MSS HEK293 cells stably expressing an anti-SIL1 shRNA display morphological alterations reminiscent of those seen in muscle fibres of MSS patients, including widened, proliferated ER, increased lysosome activity, and autophagic vacuoles ( Table 2), suggesting ER stress and altered protein clearance [36]. Proteomic analysis identified 459 differently expressed proteins between SIL1-KD and control cells, 141 of which were up-and 318 down-regulated. ...
... Proteomic analysis identified 459 differently expressed proteins between SIL1-KD and control cells, 141 of which were up-and 318 down-regulated. The proteins with altered expression were indicative of alterations to mitochondria, cytoplasm, cytoskeleton, nucleus, ER, Golgi, ERAD and secretory protein transport [36]. ...
Secretory and cell membrane proteins are synthesized in the endoplasmic reticulum (ER), where a network of molecular chaperones and folding factors ensure correct protein folding and export to post‐ER compartments. Failure of this process leads to accumulation of unfolded/misfolded proteins, ER stress, and activation of the unfolded protein response (UPR), a complex signalling pathway aimed at restoring ER homeostasis, whose failure eventually leads to cell death. SIL1 is a nucleotide exchange factor for BiP, the main ER chaperone and primary sensor of ER stress. Loss of SIL1 function causes Marinesco‐Sjögren syndrome (MSS), a rare multisystem disease of early infancy for which there is no cure. This review, examines the current understanding of SIL1 activities in the ER, and reviews experimental data describing the consequences of SIL1 deficiency in cell and animal models. We discuss the evidence supporting a role of the UPR –particularly the PERK branch – in the pathogenesis of MSS, and how this may be pharmacologically manipulated for treatment.
... SIL1 is protective against ER stress and reduces the formation of mutant SOD1 inclusions in vitro. Conversely SIL1 depletion leads to disturbed ER and nuclear envelope morphology, defective mitochondrial function, and ER stress, thus linking SIL1 to neurodegeneration (Roos et al., 2016). Furthermore, AAV-mediated overexpression of SIL1 in MNs of SOD1 G93A mice preserves FF MN axons and prolongs survival by 25-30% compared to littermates (Filézac de L'Etang et al., 2015). ...
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of both upper and lower motor neurons (MNs) in the brain, brainstem and spinal cord. The neurodegenerative mechanisms leading to MN loss in ALS are not fully understood. Importantly, the reasons why MNs are specifically targeted in this disorder are unclear, when the proteins associated genetically or pathologically with ALS are expressed ubiquitously. Furthermore, MNs themselves are not affected equally; specific MNs subpopulations are more susceptible than others in both animal models and human patients. Corticospinal MNs and lower somatic MNs, which innervate voluntary muscles, degenerate more readily than specific subgroups of lower MNs, which remain resistant to degeneration, reflecting the clinical manifestations of ALS. In this review, we discuss the possible factors intrinsic to MNs that render them uniquely susceptible to neurodegeneration in ALS. We also speculate why some MN subpopulations are more vulnerable than others, focusing on both their molecular and physiological properties. Finally, we review the anatomical network and neuronal microenvironment as determinants of MN subtype vulnerability and hence the progression of ALS.
... Although quantitative proteomic studies have been performed to investigate the UPR, proteins central to this signal transduction pathway (such as PERK, ATF6, IRE1a, CHOP, XBP1, GADD34 and ATF4) proved as notoriously difficult to detect and to quantify 3,[30][31][32] . Here we present a high resolution targeted proteomics workflow with enhanced sensitivity and selectivity for the analysis of low abundancy proteins that does not require enrichment, subcellular fractionation or two-dimensional separation steps. ...
Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.
... Native-PAGE was carried out as described in a protocol available online (http://www.assay-protocol.com/molecular-biology/ electrophoresis/native-page) and the immunoblot studies were performed as described previously (16). The following antibodies were used: ...
... Preparation of Hek293-TRex cell lines and subsequent transmission electron microscopic studies were carried out as described previously (12,16). For each SIL1 variant as well as for the wildtype protein, at least 50 cells were analyzed showing similar (patho)morphology of subcellular organelles/structures. ...
... Mitochondrial vulnerability has been repeatedly reported in MSS and woozy mouse tissues (13,(18)(19)(20), and in in vitro models of the disease (13,16). As mitochondrial function is essential for cellular fitness, we examined metabolic activity of our in vitro models by focussing on processes related to mitochondrial activity as well as on mitochondrial morphology. ...
Background and objective: Recessive mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), a rare neuropediatric disorder. MSS-patients typically present with congenital cataracts, intellectual disability, cerebellar ataxia and progressive vacuolar myopathy. However, atypical clinical presentations associated with SIL1 mutations have been described over the last years; compound heterozygosity of SIL1 missense mutations even resulted in a phenotype not fulfilling the clinical diagnostic criteria of MSS. Thus, a read-out system to evaluate reliably the pathogenicity of amino acid changes in SIL1 is needed. Here, we aim to provide suitable cellular biomarkers enabling the robust evaluation of pathogenicity of SIL1 mutations.
Methods: Five SIL1 variants including one polymorphism (p.K132Q), three known pathogenic mutations (p.V231_I232del, p.G312R, and p.L457P) and one ambiguous missense variant (p.R92W) were studied along with the wild-type proteins in Hek293 in vitro models by cell biological assays, immunoprecipitation, immunoblotting, and immunofluorescence as well as electron microscopy. Moreover, the SIL1-interactomes were interrogated by tandem-affinity-purification and subsequent mass spectrometry.
Results: Our combined studies confirmed the pathogenicity of p.V231_I232del, p.G312R, and p.L457P by showing instability of the proteins as well as tendency to form aggregates. This observation is in line with altered structure of the ER-Golgi system and vacuole formation upon expression of these pathogenic SIL1-mutants as well as the presence of oxidative or ER-stress. Reduced cellular fitness along with abnormal mitochondrial architecture could also be observed. Notably, both the polymorphic p.K132Q and the ambiguous p.R92W variants did not elicit such alterations. Study of the SIL1-interactome identified POC1A as a novel binding partner of wild-type SIL1; the interaction is disrupted upon the presence of pathogenic mutants but not influenced by the presence of benign variants. Disrupted SIL1-POC1A interaction is associated with centrosome disintegration.
Conclusions: We developed a combination of cellular outcome measures to evaluate the pathogenicity of SIL1 variants in suitable in vitro models and demonstrated that the p. R92W missense variant is a polymorphism rather than a pathogenic mutation leading to MSS.
... In the nervous system, ER stress has been identified as a key process in multiple neurodegenerative conditions such as Alzheimer's and Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS) and prion diseases (Doyle et al., 2011;Stetler et al., 2010). In this context, it is important to note that apart from the above described MSS phenotype, a prominent role of SIL1 in maintaining integrity and function of the nervous system is suggested by the following observations: (i) SIL1 has been described as a disease-modifying protein in ALS and Alzheimer's disease, respectively (Filézac de L'Etang et al., 2015;Liu et al., 2016), (ii) in vitro overexpression of SIL1 resulted in elevation of a variety of proteins with neuroprotective functions (Labisch et al., 2017), (iii) in vitro depletion of SIL1 affects several proteins important for neuronal function (Roos et al., 2016) and (iv) morphological studies of Sil1mutant mice revealed pathological alterations of nerve terminals and neuromuscular junctions along with signs of neurogenic muscular atrophy (Filézac de L'Etang et al., 2015). Although Horvers and coworkers found no clear evidence of peripheral neuropathy in their cohort of four Dutch MSS patients with proven SIL1 mutations (Horvers et al., 2013); still, the evidence discussed above suggests a vulnerability of the PNS against loss of functional SIL1. ...
... These aggregates were remarkably prominent in myelinated axons of Sil1-mutant mice and were found to be more pronounced in 26-compared to 16-week-old animals. Interestingly, abnormal aggregates of autophagic material and perturbations of nuclear envelope structures as well as mitochondrial degeneration have already been extensively described in MSS-patient and woozy mouse muscle as well as in in vitro models of the disease (Roos et al., 2014;Roos et al., 2016) suggesting that these ultra-morphological changes are a consistent feature in tissues vulnerable for SIL1 loss. Moreover, this finding is in line with the results of a study linking levels of functional BiP to the activation of autophagy as a mechanism acting towards the breakdown of protein aggregates and to axonal degeneration (Penas et al., 2011). ...
Background:
Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown.
Methods:
To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose.
Results:
Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms.
Conclusion:
Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.
... Increased levels of RCN2, an ER-luminal protein involved in Ca 2+ and redox homeostasis [27], further suggests vulnerability of proper ER-Golgi function based on p.P104L Caveolin-3 expression. Proteomic profiling is a useful approach to obtain unbiased insights into disease pathologies and related compensatory mechanisms (for example: [10,29,30]). We compared the proteomic signature of quadriceps muscles from three transgenic and wild-type littermate animals (age 10 weeks; muscles from the right legs of male animals). ...
Background
Caveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage. So far, more than 40 dominant pathogenic mutations have been described leading to several phenotypes many of which are associated with a mis-localization of the mutant protein to the Golgi. Golgi retention and endoplasmic reticulum (ER) stress has been demonstrated for the CAV3 p.P104L mutation, but further downstream pathophysiological consequences remained elusive so far. Methods
We utilized a transgenic (p.P104L mutant) mouse model and performed proteomic profiling along with immunoprecipitation, immunofluorescence and immunoblot examinations (including examination of α-dystroglycan glycosylation), and morphological studies (electron and coherent anti-Stokes Raman scattering (CARS) microscopy) in a systematic investigation of molecular and subcellular events in p.P104L caveolinopathy. ResultsOur electron and CARS microscopic as well as immunological studies revealed Golgi and ER proliferations along with a build-up of protein aggregates further characterized by immunoprecipitation and subsequent mass spectrometry. Molecular characterization these aggregates showed affection of mitochondrial and cytoskeletal proteins which accords with our ultra-structural findings. Additional global proteomic profiling revealed vulnerability of 120 proteins in diseased quadriceps muscle supporting our previous findings and providing more general insights into the underlying pathophysiology. Moreover, our data suggested that further DGC components are altered by the perturbed protein processing machinery but are not prone to form aggregates whereas other sarcolemmal proteins are ubiquitinated or bind to p62. Although the architecture of the ER and Golgi as organelles of protein glycosylation are altered, the glycosylation of α-dystroglycan presented unchanged. Conclusions
Our combined data classify the p.P104 caveolinopathy as an ER-Golgi disorder impairing proper protein processing and leading to aggregate formation pertaining proteins important for mitochondrial function, cytoskeleton, ECM remodeling and sarcolemmal integrity. Glycosylation of sarcolemmal proteins seems to be normal. The new pathophysiological insights might be of relevance for the development of therapeutic strategies for caveolinopathy patients targeting improved protein folding capacity.
