scholarly journals Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B

2020 ◽  
Vol 117 (51) ◽  
pp. 32453-32463
Author(s):  
Mafalda Concilli ◽  
Raffaella Petruzzelli ◽  
Silvia Parisi ◽  
Federico Catalano ◽  
Francesco Sirci ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Wilson Disease ◽  
Therapeutic Strategy ◽  
Copper Accumulation ◽  
Loss Of Function ◽  
Copper Transporter ◽  
Hepatic Copper ◽  
Hepatic Cells ◽  
Approved Drugs ◽  
And Function

Pathogenic mutations in the copper transporterATP7Bhave been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease.

scholarly journals Regulation of murine copper homeostasis by members of the COMMD protein family

2020 ◽  
Vol 14 (1) ◽  
pp. dmm045963
Author(s):  
Amika Singla ◽  
Qing Chen ◽  
Kohei Suzuki ◽  
Jie Song ◽  
Alina Fedoseienko ◽  
...  
Keyword(s):  
Copper Homeostasis ◽  
Copper Accumulation ◽  
Copper Transporter ◽  
Copper Excretion ◽  
Copper Absorption ◽  
Hepatic Copper ◽  
High Copper ◽  
Molecular Machinery ◽  
And Function

ABSTRACTCopper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under low- or high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Deficiency in the double-stranded RNA binding protein HYPONASTIC LEAVES1 increases sensitivity to the endoplasmic reticulum stress inducer tunicamycin in Arabidopsis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Rikako Hirata ◽  
Kei-ichiro Mishiba ◽  
Nozomu Koizumi ◽  
Yuji Iwata
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Stress Responses ◽  
Rna Binding ◽  
Transcriptional Response ◽  
Mirna Biogenesis ◽  
Loss Of Function ◽  
Wild Type ◽  
Protein Coding ◽  
And Function

Abstract Objective microRNA (miRNA) is a small non-coding RNA that regulates gene expression by sequence-dependent binding to protein-coding mRNA in eukaryotic cells. In plants, miRNA plays important roles in a plethora of physiological processes, including abiotic and biotic stress responses. The present study was conducted to investigate whether miRNA-mediated regulation is important for the endoplasmic reticulum (ER) stress response in Arabidopsis. Results We found that hyl1 mutant plants are more sensitive to tunicamycin, an inhibitor of N-linked glycosylation that causes ER stress than wild-type plants. Other miRNA-related mutants, se and ago1, exhibited similar sensitivity to the wild-type, indicating that the hypersensitive phenotype is attributable to the loss-of-function of HYL1, rather than deficiency in general miRNA biogenesis and function. However, the transcriptional response of select ER stress-responsive genes in hyl1 mutant plants was indistinguishable from that of wild-type plants, suggesting that the loss-of-function of HYL1 does not affect the ER stress signaling pathways.

scholarly journals Wilson disease: intersecting DNA methylation and histone acetylation regulation of gene expression in a mouse model of hepatic copper accumulation

2021 ◽  
Author(s):  
Gaurav V. Sarode ◽  
Kari Neier ◽  
Noreene M. Shibata ◽  
Yuanjun Shen ◽  
Dmitry A Goncharov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Mouse Model ◽  
Histone Acetylation ◽  
Wilson Disease ◽  
Metabolic Pathways ◽  
Histone Deacetylases ◽  
Copper Accumulation ◽  
Copper Transporter ◽  
Pathogenic Variants

AbstractThe pathogenesis of Wilson disease (WD) is multi-factorial, involving hepatic and brain copper accumulation due to pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior DNA methylation investigations in human WD liver and blood and in a WD mouse model revealed an epigenetic signature of WD, including alterations in the histone deacetylase HDAC5. To test the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD, we investigated class IIa histone deacetylases (HDAC4 and HDAC5) and H3K9/H3K27 histone acetylation in the Jackson Laboratory toxic milk (tx-j) mouse model of WD compared to C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet (HFD), D-penicillamine (PCA, copper chelator), and choline (methyl group donor). HDAC5 levels significantly increased in 9-week tx-j livers after 8 days of HFD compared to chow. In 24-week tx-j livers, HDAC4/5 levels were reduced 5- to 10-fold compared to C3H likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were also affected by disease progression and accompanied by increased acetylation. PCA and choline partially restored HDAC4, HDAC5, H3K9ac, and H3K27ac levels to that of CH3 liver. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development were, in turn, regulated by histone acetylation in tx-j mice compared to C3H, with Pparα and Pparγ among the most relevant targets. These results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation, can regulate gene expression in key metabolic pathways in the pathogenesis of WD.Significance StatementWilson disease is considered a monogenic disease caused by pathogenic variants in the ATP7B copper transporter, resulting in hepatic and brain copper accumulation. Given the lack of genotype-phenotype correlation, evidence of epigenetic and metabolic mechanisms regulating phenotype in patients and in animal models could explain the high phenotype variability observed in WD. In this study, we identify class IIa histone deacetylases as players involved in the epigenetic regulation of key metabolic pathways that can affect WD severity as well as targets sensitive to dietary modulations, which is an important characteristic for designing effective and feasible therapies. Understanding the epigenetic mechanisms in WD pathogenesis contributes to a better understanding of the phenotypic variability in WD and other common liver conditions.

Transient Elastography to Represent Hepatic Copper Accumulation in Wilson Disease

2020 ◽  
Vol 57 (8) ◽  
pp. 762-763
Author(s):  
Jirakorn Jamrasnaradom ◽  
Palittiya Sintusek
Keyword(s):  
Wilson Disease ◽  
Transient Elastography ◽  
Copper Accumulation ◽  
Hepatic Copper

scholarly journals The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis

Blood ◽  
2020 ◽  
Vol 136 (6) ◽  
pp. 715-725 ◽  
Author(s):  
Richard W. Lo ◽  
Ling Li ◽  
Fred G. Pluthero ◽  
Richard Leung ◽  
Koji Eto ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Dynamic Network ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Function Loss ◽  
Endoplasmic Reticulum Protein ◽  
Gray Platelet Syndrome ◽  
Endogenous Proteins ◽  
And Function

Abstract Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes gray platelet syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Coimmunoprecipitation (co-IP) experiments using tagged proteins expressed in human HEK293 and megakaryocytic immortalized megakaryocyte progenitor (imMKCL) cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-IP, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798 to 1903, where 2 GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B coexpressed in HEK293 cells. CRISPR/Cas9-mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.

scholarly journals Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat

1994 ◽  
Vol 301 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Y Yamaguchi ◽  
M E Heiny ◽  
N Shimizu ◽  
T Aoki ◽  
J D Gitlin
Keyword(s):  
Wilson Disease ◽  
Molecular Basis ◽  
Disease Gene ◽  
Genetic Disorder ◽  
Copper Accumulation ◽  
Hepatic Copper ◽  
Examine Gene Expression ◽  
Bona Fide ◽  
Rat Strain ◽  
Long Evans

Long-Evans Cinnamon rats develop a necrotizing hepatitis characterized by excessive hepatic copper accumulation, defective holoceruloplasmin biosynthesis and impaired biliary copper excretion. To elucidate the molecular basis of this defect, a cDNA clone encoding the rat Wilson disease gene was isolated and used to examine gene expression in selected tissues from normal and Long-Evans Cinnamon rats. Although this cDNA readily detects Wilson transcripts in liver and other tissues from normal rats, such transcripts are entirely absent from tissues derived from the Long-Evans Cinnamon rat strain. These data therefore identify the Long-Evans Cinnamon rat as the first bona fide animal model of Wilson disease and suggest that this rat strain may be a valuable resource in the study of this genetic disorder.

scholarly journals Neuronal dystonin isoform 2 is a mediator of endoplasmic reticulum structure and function

2012 ◽  
Vol 23 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Scott D. Ryan ◽  
Andrew Ferrier ◽  
Tadasu Sato ◽  
Ryan W. O'Meara ◽  
Yves De Repentigny ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Sensory Neurons ◽  
Stress Proteins ◽  
Function Analysis ◽  
Structure And Function ◽  
Loss Of Function ◽  
Specific Loss ◽  
And Function

Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt27 mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca2+ dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.

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