Accumulation Of Mutant Protein Research Articles

A human-specific cytotoxic neopeptide generated by the deafness gene Cingulin

Accumulation of mutant proteins in cells can induce proteinopathies and cause functional damage to organs. Recently, the Cingulin (CGN) protein has been shown to maintain the morphology of cuticular plates of inner ear hair cells and a frameshift mutation in CGN causes autosomal dominant nonsyndromic hearing loss. Here, we find that the mutant CGN proteins form insoluble aggregates which accumulate intracellularly and lead to cell death. Expression of the mutant CGN in the inner ear results in severe hair cell death and hearing loss in mice, resembling the auditory phenotype in human patients. Interestingly, a human-specific residue (V1112) in the neopeptide generated by the frameshift mutation is critical for the aggregation and cytotoxicity of the mutant human CGN. Moreover, the expression of heat shock factor 1 (HSF1) decreases the accumulation of insoluble mutant CGN aggregates and rescues cell death. In summary, these findings identify mutant-specific toxic polypeptides as a disease-causing mechanism of the deafness mutation in CGN, which can be targeted by the expression of the cell chaperone response regulator HSF1.

A method for the analysis of the oligomerization profile of the Huntington's disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation.

Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.

Aggregation of E121K mutant D-amino acid oxidase and ubiquitination-mediated autophagy mechanisms leading to amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a terminal adult-onset neuromuscular disorder. Our group has been studying this illness and previously reported novel mutations and rare mutations in a study using next-generation sequencing of DNA samples from Indian ALS patients. In this paper, we focus on the E121K mutation in the DAO gene to understand how it leads to ALS. Our experiments in SH-SY5Y cells indicate that the E121K mutation results in the accumulation of mutant protein aggregates, a change in cell morphology, and the death of neuronal cells. These protein aggregates get ubiquitinated and cause an imbalance in autophagy regulation. We observed an increase in the cellular concentrations of p62, OPTN, and LC3II. Through confocal microscopy studies, we show that the binding of p62 with ubiquitinated aggregates and its recruitment to LC3II mediates autophagosome generation. These relative changes in the key partners in autophagy increase cell death in cells harboring the E121K mutation and is a probable mechanism leading to ALS.

A Specific Mini-Intrabody Mediates Lysosome Degradation of Mutant Huntingtin.

Accumulation of misfolded proteins leads to many neurodegenerative diseases that can be treated by lowering or removing mutant proteins. Huntington's disease (HD) is characterized by the intracellular accumulation of mutant huntingtin (mHTT) that can be soluble and aggregated in the central nervous system and causes neuronal damage and death. Here, an intracellular antibody (intrabody) fragment is generated that can specifically bind mHTT and link to the lysosome for degradation. It is found that delivery of this peptide by either brain injection or intravenous administration can efficiently clear the soluble and aggregated mHTT by activating the lysosomal degradation pathway, resulting in amelioration of gliosis and dyskinesia in HD knock-in (KI-140Q) mice. These findings suggest that the small intrabody peptide linked to lysosomes can effectively lower mutant proteins and provide a new approach for treating neurodegenerative diseases that are caused by the accumulation of mutant proteins.

Functional characterization of a rare pathogenic variant c.875G>A, p.(Cys292Tyr) in COMP.

The protein encoded by the cartilage oligomeric matrix protein (COMP) gene is a noncollagenous extracellular matrix (ECM) protein that is important for chondrocyte formation and growth. Variations in the COMP gene cause pseudoachondroplasia (PSACH), which is mainly characterized by short-limbed dwarfing in the clinic. To characterize the function of a rare pathogenic variant in the COMP gene (c.875G>A, p.Cys292Tyr). We performed 3D structural analysis, in vitro expression analysis, and immunofluorescence to characterize the effects of the variant on protein structure, expression, and cellular localization respectively. Variation modeling showed that the interactions between amino acids were changed after the variation, and there were 31 changes in the secondary structure of mutant COMP (MT-COMP). Western blot showed that the intracellular quantity of MT-COMP was higher than the wild-type COMP (WT-COMP). Cellular immunofluorescence results showed that WT-COMP was less abundant and homogenously distributed in cells, while the MT-COMP accumulated in the cytoplasm. Herein, we report a variant of COMP in a Chinese family with PSACH. We have shown that the rare missense variant, COMP c.875G>A, previously reported in ClinVar and identified in our patient, results in excessive accumulation of mutant protein in the cytoplasm, and is therefore pathogenic. Through in silico and experimental analyses, we provide evidence that COMP c.875G>A is the likely cause of PSACH in a Chinese family.

Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease.

Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, we expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, we examined the contribution of autophagy in mitigating toxicity caused by mutant HTT protein. We found that targeted overexpression of an autophagy gene, Atg8a in male flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, we demonstrate that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. We show that the rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, our study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.SIGNIFICANCE STATEMENT Defects in sleep and circadian rhythms are well documented in Huntington's disease. Recent literature suggests that circadian and sleep disturbances can exacerbate neurodegenerative phenotypes. Hence, identifying potential modifiers that can improve the functioning of these circuits could greatly improve disease management. We used a genetic approach to enhance cellular proteostasis and found that overexpression of a crucial autophagy gene, Atg8a, induces the autophagy pathway in the Drosophila circadian and sleep neurons and rescues sleep and activity rhythm. We demonstrate that the Atg8a improves synaptic function of these circuits by possibly enhancing the aggregation of the mutant protein in neurons. Further, our results suggest that differences in basal levels of protein homeostatic pathways is a factor that determines selective susceptibility of neurons.

Rare protective variants and glaucoma-relevant cell stressors modulate Angiopoietin-like 7 expression.

Rare missense and nonsense variants in the Angiopoietin-like 7 (ANGPTL7) gene confer protection from primary open-angle glaucoma (POAG), though the functional mechanism remains uncharacterized. Interestingly, a larger variant effect size strongly correlates with in silico predictions of increased protein instability (r = -0.98), suggesting that protective variants lower ANGPTL7 protein levels. Here, we show that missense and nonsense variants cause aggregation of mutant ANGPTL7 protein in the endoplasmic reticulum (ER) and decreased levels of secreted protein in human trabecular meshwork (TM) cells; a lower secreted:intracellular protein ratio strongly correlates with variant effects on intraocular pressure (r = 0.81). Importantly, accumulation of mutant protein in the ER does not increase expression of ER stress proteins in TM cells (P > 0.05 for all variants tested). Cyclic mechanical stress, a glaucoma-relevant physiologic stressor, also significantly lowers ANGPTL7 expression in primary cultures of human Schlemm's canal (SC) cells (-2.4-fold-change, P = 0.01). Collectively, these data suggest that the protective effects of ANGPTL7 variants in POAG stem from lower levels of secreted protein, which may modulate responses to physiologic and pathologic ocular cell stressors. Downregulation of ANGPTL7 expression may therefore serve as a viable preventative and therapeutic strategy for this common, blinding disease.

Cytoplasmic aggregation of mutant FUS causes multistep RNA splicing perturbations in the course of motor neuron pathology.

Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.

The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein.

Conformational diseases, such as Alzheimer, Parkinson and Huntington diseases, are part of a common class of neurological disorders characterized by the aggregation and progressive accumulation of proteins bearing aberrant conformations. Huntington disease (HD) has autosomal dominant inheritance and is caused by mutations leading to an abnormal expansion in the polyglutamine (polyQ) tract of the huntingtin (HTT) protein, leading to the formation of HTT inclusion bodies in neurons of affected patients. Interestingly, recent experimental evidence is challenging the conventional view by which the disease pathogenesis is solely a consequence of the intracellular accumulation of mutant protein aggregates. These studies reveal that transcellular transfer of mutated huntingtin protein is able to seed oligomers involving even the wild-type (WT) forms of the protein. To date, there is still no successful strategy to treat HD. Here, we describe a novel functional role for the HSPB1-p62/SQSTM1 complex, which acts as a cargo loading platform, allowing the unconventional secretion of mutant HTT by extracellular vesicles. HSPB1 interacts preferentially with polyQ-expanded HTT compared with the WT protein and affects its aggregation. Furthermore, HSPB1 levels correlate with the rate of mutant HTT secretion, which is controlled by the activity of the PI3K/AKT/mTOR signalling pathway. Finally, we show that these HTT-containing vesicular structures are biologically active and able to be internalized by recipient cells, therefore providing an additional mechanism to explain the prion-like spreading properties of mutant HTT. These findings might also have implications for the turn-over of other disease-associated, aggregation-prone proteins.

Cell Rearrangement and Oxidant/Antioxidant Imbalance in Huntington's Disease.

Huntington's Disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a CAG triplet repeat in the HTT gene, resulting in the production of an aberrant huntingtin (Htt) protein. The mutant protein accumulation is responsible for neuronal dysfunction and cell death. This is due to the involvement of oxidative damage, excitotoxicity, inflammation, and mitochondrial impairment. Neurons naturally adapt to bioenergetic alteration and oxidative stress in physiological conditions. However, this dynamic system is compromised when a neurodegenerative disorder occurs, resulting in changes in metabolism, alteration in calcium signaling, and impaired substrates transport. Thus, the aim of this review is to provide an overview of the cell's answer to the stress induced by HD, focusing on the role of oxidative stress and its balance with the antioxidant system.

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of β-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630–1640 cm-1, attributed to a parallel β-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.

Response to endoplasmic reticulum stress in arginine vasopressin neurons.

Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Biochemical Pathways of Cellular Mechanosensing/Mechanotransduction and Their Role in Neurodegenerative Diseases Pathogenesis.

In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with particular attention to their role in neurodegenerative disease pathogenesis. While the mechanistic components of these pathways are mostly uncovered today, the crosstalk between mechanical forces and soluble intracellular signaling is still not fully elucidated. Here, we recapitulate the general concepts of mechanobiology and the mechanisms that govern the mechanosensing and mechanotransduction processes, and we examine the crosstalk between mechanical stimuli and intracellular biochemical response, highlighting their effect on cellular organelles’ homeostasis and dysfunction. In particular, we discuss the current knowledge about the translation of mechanosignaling into biochemical signaling, focusing on those diseases that encompass metabolic accumulation of mutant proteins and have as primary characteristics the formation of pathological intracellular aggregates, such as Alzheimer’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis and Parkinson’s Disease. Overall, recent findings elucidate how mechanosensing and mechanotransduction pathways may be crucial to understand the pathogenic mechanisms underlying neurodegenerative diseases and emphasize the importance of these pathways for identifying potential therapeutic targets.

In Vitro Efficacy and Molecular Mechanism of Curcumin Analog in Pathological Regulation of Spinocerebellar Ataxia Type 3.

Unlike other nuclear factor erythroid-2-related factor 2 (Nrf2) activators, the mechanism of action of curcumin analog, ASC-JM17 (JM17), in regulating oxidative homeostasis remains unknown. Spinocerebellar ataxia type 3 (SCA3) is an inherited polyglutamine neurodegenerative disease caused mainly by polyglutamine neurotoxicity and oxidative stress. Presently, we compared actions of JM17 with those of known Nrf2 activators, omaveloxolone (RTA-408) and dimethyl fumarate (DMF), using human neuroblastoma SK-N-SH cells with stable transfection of full-length ataxin-3 protein with 78 CAG repeats (MJD78) to clarify the resulting pathological mechanism by assaying mitochondrial function, mutant ataxin-3 protein toxicity, and oxidative stress. JM17, 1 μM, comprehensively restored mitochondrial function, decreased mutant protein aggregates, and attenuated intracellular/mitochondrial reactive oxygen species (ROS) levels. Although JM17 induced dose-dependent Nrf2 activation, a low dose of JM17 (less than 5 μM) still had a better antioxidant ability compared to the other Nrf2 activators and specifically increased mitochondrial superoxide dismutase 2 in an Nrf2-dependent manner as shown by knockdown experiments with siRNA. It showed that activation of Nrf2 in response to ROS generated in mitochondria could play an import role in the benefit of JM17. This study presents the diversified regulation of JM17 in a pathological process and helped develop more effective therapeutic strategies for SCA3.

Behavioral- and blood-based biomarkers for Huntington's disease: Studies on the R6/1 mouse model with prospects for early diagnosis and monitoring of the disease

BackgroundHuntington's disease (HD) is a genetic neurodegenerative disorder characterized by a triad of cognitive, psychiatric and motor symptoms. One of the main mechanisms of the disease, besides the aggregation of mutant proteins, is the chronic inflammation that occurs in patients long before the onset of motor dysfunction. Currently, no effective therapy is available for HD. As the progression of HD is usually slow and its first clinical manifestations are non-specific, a correct diagnosis is difficult. Moreover, any attempts to develop therapies for HD require monitoring of unequivocal markers of the disease which are also hardly available. ObjectiveUsing a mouse model of HD, we aimed to determine a battery of biomarkers in peripheral blood, as well as those related to cognitive or anxiety disturbances, corresponding to the most characteristic breakpoints in HD. MethodsThe R6/1 mouse line was used as an animal HD model. Levels of cytokines and hormones in blood were estimated by ELISA. A series of behavioral and movement tests was applied. ResultsSignificant elevation of levels of specific inflammatory markers (IL-6, TNF-α, IL-1β, IL-12) was observed in the course of HD. At early stages of the disease, an impairment of anti-inflammatory defense mechanisms was evident, expressed by a significant decrease in IL-10 levels. The disturbances were faster in females than in males which might also translate into a potentially worse response to any administered drugs. Impaired memory and anxiety could be detected. ConclusionA set of simple behavioral- and blood-based biomarkers for HD has been detected.

A peptide inhibitor that rescues polyglutamine-induced synaptic defects and cell death through suppressing RNA and protein toxicities

Polyglutamine (polyQ) diseases, including spinocerebellar ataxias and Huntington’s disease, are progressive neurodegenerative disorders caused by CAG triplet-repeat expansion in the coding regions of disease-associated genes. In this study, we found that neurotoxic small CAG (sCAG) RNA species, microscopic Ataxin-2 CAG RNA foci, and protein aggregates exist as independent entities in cells. Synaptic defects and neurite outgrowth abnormalities were observed in mutant Ataxin-2-expressing mouse primary cortical neurons. We examined the suppression effects of the CAG RNA-binding peptide beta-structured inhibitor for neurodegenerative diseases (BIND) in mutant Ataxin-2-expressing mouse primary cortical neurons and found that both impaired synaptic phenotypes and neurite outgrowth defects were rescued. We further demonstrated that BIND rescued cell death through inhibiting sCAG RNA production, Ataxin-2 CAG RNA foci formation, and mutant Ataxin-2 protein translation. Interestingly, when the expanded CAG repeats in the mutant Ataxin-2 transcript was interrupted with the alternative glutamine codon CAA, BIND’s inhibitory effect on mutant protein aggregation was lost. We previously demonstrated that BIND interacts physically and directly with expanded CAG RNA sequences. Our data provide evidence that the BIND peptide associates with transcribed mutant CAG RNA to inhibit the formation of toxic species, including sCAG RNA, RNA foci, and polyQ protein translation and aggregation.

Exploring the Role of Posttranslational Modifications in Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked adult-onset progressive neuromuscular disease that affects the spinal and bulbar motor neurons and skeletal muscles. SBMA is caused by expansion of polymorphic CAG trinucleotide repeats in the Androgen Receptor (AR) gene, resulting in expanded glutamine tract in the AR protein. Polyglutamine (polyQ) expansion renders the mutant AR protein toxic, resulting in the formation of mutant protein aggregates and cell death. This classifies SBMA as one of the nine known polyQ diseases. Like other polyQ disorders, the expansion of the polyQ tract in the AR protein is the main genetic cause of the disease; however, multiple other mechanisms besides the polyQ tract expansion also contribute to the SBMA disease pathophysiology. Posttranslational modifications (PTMs), including phosphorylation, acetylation, methylation, ubiquitination, and SUMOylation are a category of mechanisms by which the functionality of AR has been found to be significantly modulated and can alter the neurotoxicity of SBMA. This review summarizes the different PTMs and their effects in regulating the AR function and discusses their pathogenic or protective roles in context of SBMA. This review also includes the therapeutic approaches that target the PTMs of AR in an effort to reduce the mutant AR-mediated toxicity in SBMA.

Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation.

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β-blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation.

Novel NPR2 Gene Mutations Affect Chondrocytes Function via ER Stress in Short Stature.

Natriuretic peptide receptor 2 (NPR2) plays a key role in cartilage and bone morphogenesis. The NPR2 gene mutations result in acromesomelic dysplasia, Maroteaux type (AMDM), short stature with nonspecific skeletal abnormalities (SNSK), and epiphyseal chondrodysplasia, Miura type (ECDM). However, the pathogenic mechanism remains unclear. In our study, we identified one de novo (R557C) and six novel variants (G602W, V970F, R767*, R363*, F857S, and Y306S) in five independent Chinese families with familial short stature. Three patients with heterozygous mutations (G602W, V970F, and R767*) were diagnosed with SNSK (height SD score ranged from −2.25 to −5.60), while another two with compound heterozygous mutations (R363* and F857S, R557C and Y306S) were diagnosed with AMDM (height SD score ranged from −3.10 to −5.35). Among three patients with heterozygous status, two patients before puberty initiation with rhGH treatment significantly improved their growth (height velocity 7.2 cm/year, 6.0 cm/year), and one patient in puberty had a poor response to the rhGH treatment (height velocity 2.5 cm/year). Seven NPR2 gene variants were constructed and overexpressed in HEK293T and ATDC5 cells, and we found that ATDC5 cells with mutant NPR2 gene showed decreased differentiation, as evidenced by lower expression of ColII, ColX, and BMP4 and higher expression of Sox9. Moreover, the apoptosis rate was elevated in ATDC5 cells expressing the mutant NPR2 gene. N-glycosylation modification, plasma membrane localization, and ER stress resulted from the accumulation of mutant protein in ER, as shown by the higher expression of GRP78 and p-IRE1α. Overall, our results provide a novel insight into NPR2 loss of function, which could promote chondrocyte apoptosis and repress cell differentiation through ER stress and the unfolded protein response.