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Background: Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles made of hyperphosphorylated tau and senile plaques composed of beta-amyloid. These pathognomonic deposits have been implicated in the pathogenesis, although the molecular mechanisms and consequences remain undetermined. UFM1 is an important, but understudied ubiquitin-like protein that is covalently attached to substrates. This UFMylation has recently been identified as major modifier of tau aggregation upon seeding in experimental models. However, potential alterations of the UFM1 pathway in human AD brain have not been investigated yet. Methods: Here we used frontal and temporal cortex samples from individuals with or without AD to measure the protein levels of the UFMylation pathway in human brain. We used multivariable regression analyses followed by Bonferroni correction for multiple testing to analyze associations of the UFMylation pathway with neuropathological characteristics, primary biochemical measurements of tau and additional biochemical markers from the same cases. We further studied associations of the UFMylation cascade with cellular stress pathways using Spearman correlations with bulk RNAseq expression data and functionally validated these interactions using gene-edited neurons that were generated by CRISPR-Cas9. Results: Compared to controls, human AD brain had increased protein levels of UFM1. Our data further indicates that this increase mainly reflects conjugated UFM1 indicating hyperUFMylation in AD. UFMylation was strongly correlated with pathological tau in both AD-affected brain regions. In addition, we found that the levels of conjugated UFM1 were negatively correlated with soluble levels of the deUFMylation enzyme UFSP2. Functional analysis of UFM1 and/or UFSP2 knockout neurons revealed that the DNA damage response as well as the unfolded protein response are perturbed by changes in neuronal UFM1 signaling. Conclusions: There are marked changes in the UFMylation pathway in human AD brain. These changes are significantly associated with pathological tau, supporting the idea that the UFMylation cascade might indeed act as a modifier of tau pathology in human brain. Our study further nominates UFSP2 as an attractive target to reduce the hyperUFMylation observed in AD brain but also underscores the critical need to identify risks and benefits of manipulating the UFMylation pathway as potential therapeutic avenue for AD.
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Spondyloepimetaphyseal dysplasia (SEMD) is characterized by vertebral, epiphyseal, and metaphyseal alterations. Patients become predominantly apparent with disproportionate short stature. The genetic background of SEMD is heterogeneous, with different modes of inheritance (autosomal dominant, autosomal recessive, and X-linked disorders). Amongst the genes in which variants are known to cause SEMD, UFM1-specific protease 2 (UFSP2) encodes a cysteine protease involved in the maturation of Ubiquitin-fold modifier 1 (UFM1). Heterozygous pathogenic variants affecting the C-terminal catalytic domain of UFSP2 are related to two entities of skeletal dysplasia, Beukes hip dysplasia (BHD) and SEMD type Di Rocco (SEMDDR). This is the first report of a de novo heterozygous variant affecting the catalytic Cys302 residue of UFSP2 (NM_018359.3:c.905G>C, p.(Cys302Ser)) causing SEMDDR. According to previously described patients with SEMDDR, our patient presented with disproportionate short stature, genu varum, gait instability, and radiologically detected epiphyseal and metaphyseal alterations. Additionally, a bell-shaped thorax, lumbar hyperlordosis, muscular hypotonia, and coxa vara were observed in the patient described in this study. Our findings underline the fundamental importance of an intact catalytic triad of the human UFSP2 for normal skeletal development and extend the phenotypical features of patients with UFSP2-related skeletal dysplasia.
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The ufmylation ligase-UFL1 promotes ATM activation by monoufmylating H4 at K31 in a positive-feedback loop after double-strand breaks (DSB) occur, whereas UFM1 Specific Peptidase 2 (UfSP2) suppresses ATM activation, but the mechanism of recruitment of UfSP2 to the DSB finetuning DNA damage response is still not clear. Here, we report that UfSP2 foci formation is delayed compared to UFL1 foci formation following the radiation insult. Mechanistically, UfSP2 binds to the MRN complex in absence of DSB. Irradiation-induced phosphorylation of UfSP2 by ATM leads to the dissociation of UfSP2 from the MRN complex. This phosphorylation can be removed by the phosphatase WIP1, thereby UfSP2 is recruited to the DSBs, deufmylating H4 and suppressing ATM activation. In summary, we identify a mechanism of delicately negative modulation of ATM activation by UfSP2 and rewires ATM activation pathways.
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Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like posttranslational modification with important biological functions. However, the regulatory mechanisms governing UFM1 modification of target proteins (UFMylation) and the cellular processes controlled by UFMylation remain largely unknown. It has been previously shown that a UFM1-specific protease (UFSP2) mediates the maturation of the UFM1 precursor and drives the de-UFMylation reaction. Furthermore, it has long been thought that UFSP1, an ortholog of UFSP2, is inactive in many organisms, including human, because it lacks an apparent protease domain when translated from the canonical start codon (445AUG). Here, we demonstrate using the combination of site-directed mutagenesis, CRISPR/Cas9-mediated genome editing, and mass spectrometry approaches that translation of human UFSP1 initiates from an upstream near-cognate codon, 217CUG, via eukaryotic translation initiation factor eIF2A-mediated translational initiation rather than from the annotated 445AUG, revealing the presence of a catalytic protease domain containing a Cys active site. Moreover, we show that both UFSP1 and UFSP2 mediate maturation of UFM1 and de-UFMylation of target proteins. This study demonstrates that human UFSP1 functions as an active UFM1-specific protease, thus contributing to our understanding of the UFMylation/de-UFMylation process.
Assuntos
Cisteína Endopeptidases , Peptídeo Hidrolases , Proteínas , Códon de Iniciação/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Humanos , Peptídeo Hidrolases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Ubiquitina/metabolismoRESUMO
Silicosis is an irreversible occupational pulmonary disease that is characterized as progressed pulmonary fibrosis. In this study, we investigated the changes of UFSP2 and the related UFMylation in silica-induced pulmonary injury mice models. The experimental silicosis models were prepared by intratracheal injection of silica particles, and the lung samples were harvested at the first or the seventh day after treatment. We found that the UFSP2 expression in the 1-day models was comparable, whereas it was upregulated in the 7-day models. Consistently, the UFMylation in the lung tissues of the 7-day models was activated. In addition, we observed the CADM2, an adhesion molecule, was reported to associate with epithelial-mesenchymal transition, was upregulated in the lungs of 7-day models. In contrast, it remained comparable in the 1-day models. Our data indicated that the UFSP2/UFMylation pathway and the CADM2 might be involved in the silica-induced pulmonary injury.
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Cisteína Endopeptidases/metabolismo , Proteínas/metabolismo , Silicose/metabolismo , Animais , China , Cisteína Endopeptidases/fisiologia , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Dióxido de Silício/efeitos adversos , Silicose/fisiopatologiaRESUMO
Spondyloepimetaphyseal dysplasias (SEMDs), which comprise a heterogeneous group of autosomal-dominant, autosomal-recessive and X-linked recessive disorders, are characterized by anomalies of the spine, the epiphyses and metaphyses of the long bones, resulting in short stature and osteoarthritic changes of the joints. UFSP2 gene encodes a highly conserved cysteine protease which cleaves two C-terminal residues from ubiquitin-fold modifier 1, an ubiquitin-like post-translational modifier protein. In 2018, Di Rocco, M reported for the first time that a novel heterozygous variant exon 11: c.1277A > C of the UFSP2 gene was the cause to spondyloepimetaphyseal dysplasia mainly manifested as: short stature, anterior vertebral dysplasia, hip dysplasia, flat vertebra, spinal metaphyseal dysplasia, irregular acetabular apex, distal femoral metaphyseal dysplasia, proximal tibial metaphyseal dysplasia, osteoarthritis and so on. In this report, we describe a boy with spondyloepimetaphyseal dysplasia due to a novel mutation exon 11: c.1283A > G (leading to p. H428R) of the UFSP2 gene. This is the second report to describe children with SEMDs associated with an UFSP2 variant. However, it is the first to describe a UFSP2 gene mutation exon 11: c.1283A > G (leading to p. H428R). Our findings of a novel heterozygous mutation of UFSP2 gene add to the list of 2 reported heterozygous mutations of UFSP2 which led to hereditary osteopathy.
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Cisteína Endopeptidases/genética , Osteocondrodisplasias/genética , Criança , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Osteocondrodisplasias/patologiaRESUMO
Beukes hip dysplasia is an autosomal dominant disease which has to date been described only in a large South African family of Dutch origin. The patients presented with progressive epiphyseal dysplasia limited to femoral capital epiphysis and their height was not significantly reduced. A unique variant of the ubiquitin-fold modifier 1 (Ufm1)-specific peptidase 2 (UFSP2) gene (c.868T>C) has been reported in all individuals from Beukes family with clinical and radiological diagnosis of Beukes hip dysplasia. Three individuals, propositus, mother, and grandmother, presented with short stature, joint pain, genu vara and a novel spondyloepimetaphyseal dysplasia involving epiphyses predominantly at hips, but also at knees, ankles, wrists and hands, associated with variable degrees of metaphysis and spine involvement. Exome sequencing allowed us to identify the heterozygous variant c.1277A>C of the UFSP2 gene, leading to the missense change p.D426A, in all 3 patients. This mutation is predicted as damaging and, similarly to the mutation originally described in the Beukes family (p. Y290H), directly affects one of the catalytic residues participating in the active site of the protein. This supports the novel notion that loss of catalytic UFSP2 activity, observed in association with different mutants and already experimentally proven in vitro, may have different clinical outcomes.