Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B−/−) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in autophagy pathway, so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which bind to PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression and cell viability shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.

A Rare Case of Wilson’s Disease in a 17 Years Old Girl

Introduction: Wilson’s disease (WD) is an autosomal recessive disorder involving cellular copper transport. A defect in biliary excretion leads to accumulation of copper in the liver, causing progressive liver injury and cirrhosis. Approximately 1 in 40,000 people have Wilson’s Disease. It affects both men and women equally. Symptoms appear between ages 5 and 35. Case Presentation: This is a case of 17 years old girl came with complained of dysphagia, irritability, breathlessness, weakness in both upper and lower limb at left side for 6 months. She had difficulty in going up and coming down a staircase and in getting up from the squatting position. She could walk with support for the first 6 months of his illness but later, had required crutches. After her admission diagnostic evaluation was done, and in the findings was chest x-ray was done which was normal. ECG showing normal sinus rate and rhythm. Ophthalmology call was done and slit lamp examination was noted which was showing KF ring was present bilaterally. MRI brain suggested symmetrical altered signal intensity lesion in bilateral thalami and lentiform nucleus, midbrain and pons appearing hyper intense on T2/FLAIR sequences in bilateral gangliocapsular region (mainly in putamen) and thalami as well as midbrain and dorsal aspect of pons as described above. A possibility of Wilson’s Disease can be considered, blood test show that Hb was decrease that is -9.7gm%, S.G.O.T was 34.0U/L, S.G.P.T was 32.0U/L, was normal at the time of discharge early ambulation, nutrition, psychological support was given. Therapeutic interventions and outcome: In the present case received syndopa 110/4, BD, orally. Tablet Zinc, OD, orally. Tablet Pan D, OD, orally., inj. Dexamethasone 2 mg I.V, T.D.S., Syrup Sumax, 10ml, BD. Now the patient condition is stable. Conclusion: This presented case of Wilson’s Disease is a rare disease condition. It is an autosomal recessive disease.In this case study, author mainly focus on expert medical management and excellent nursing care which leads to fast recovery of patient. After conversation with patient her response was positive and after nursing management and treatment she was discharged with satisfaction of recovery.

Peptides Derived from Angiogenin Regulate Cellular Copper Uptake

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes’ geometry to modulate peptides’ activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.

Role of Copper on Mitochondrial Function and Metabolism

Copper is essential for life processes like energy metabolism, reactive oxygen species detoxification, iron uptake, and signaling in eukaryotic organisms. Mitochondria gather copper for the assembly of cuproenzymes such as the respiratory complex IV, cytochrome c oxidase, and the antioxidant enzyme superoxide dismutase 1. In this regard, copper plays a role in mitochondrial function and signaling involving bioenergetics, dynamics, and mitophagy, which affect cell fate by means of metabolic reprogramming. In mammals, copper homeostasis is tightly regulated by the liver. However, cellular copper levels are tissue specific. Copper imbalances, either overload or deficiency, have been associated with many diseases, including anemia, neutropenia, and thrombocytopenia, as well as tumor development and cancer aggressivity. Consistently, new pharmacological developments have been addressed to reduce or exacerbate copper levels as potential cancer therapies. This review goes over the copper source, distribution, cellular uptake, and its role in mitochondrial function, metabolic reprograming, and cancer biology, linking copper metabolism with the field of regenerative medicine and cancer.

PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which similarly to the junctional protein PLEKHA7 bind to PDZD11 N-terminus through their WW domains. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable copper with the labile copper probe CF4 shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's Ataxia

Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. An underappreciated source of damage to Fe-S clusters are cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ to support copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3-mediated Cu1+ toxicity is a major determinant of cellular Fe-S cluster quotient. Inadequate Fe-S cluster supply, either due to diminished assembly as occurs in Friedreich's Ataxia or defective distribution, causes severe metabolic and growth defects in S. cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster-dependent metabolism and growth. Our findings reveal a novel interplay between chromatin and mitochondria in Fe-S cluster homeostasis, and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.