Identification of a novel multiprotein complex in cargo sorting that preserves metabolic pathways in the liver

Scaffold proteins are crucial regulators of a diverse array of biological processes. This thesis aims to better understand the function of the relatively new family of scaffold proteins called the COMMD proteins. COMMD1, the prototype of this family, has been associated with numerous diseases such as hepatic copper toxicity syndrome, hypercholesterolemia and cancer, but the biological role of the other nine members remains largely unknown.Our study show for the first time that in the liver COMMD6 and COMMD9 both an important role have to preserve cholesterol and copper homeostasis, similarly as we previously demonstrated for COMMD1. Interestingly, however, our data indicate that only myeloid COMMD1 prevents uncontrolled inflammation but not COMMD6 and COMMD9. We show that the COMMD proteins form together a stable multi-COMMD protein complex to regulate these cellular processes. The organization of this complex is likely cell type-specific, but the exact composition of these complexes remains unclear, and more research is wanted. Taken together our work revealed that the COMMD proteins likely act together to facilitate the endosomal trafficking of different transmembrane proteins such as LDLR and ATP7B to preserve cholesterol and copper homeostasis. We expect that better understanding of these pathways will advance therapeutic research to treat hypercholesterolemia and copper disorders

Regulation of copper homeostasis by members of the COMMD protein family

Copper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominatly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be actively recycled 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 mice with liver-specific Commd1 deficiency recapitulating aspects of this phenotype as well. 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-deficient mice. We find that in contrast to effects in cell lines in culture, COMMD protein deficiency induces minimal changes in ATP7A in enterocytes and does not lead to altered copper levels under low or high copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiologic consequence. In contrast, deficiency of any of 3 Commd genes (Commd1, 6, and 9) all result in hepatic copper accumulation under high copper diets. We find that each of these deficiencies cause 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

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation

Identification of a novel multiprotein complex in cargo sorting that preserves metabolic pathways in the liver

Scaffold proteins are crucial regulators of a diverse array of biological processes. This thesis aims to better understand the function of the relatively new family of scaffold proteins called the COMMD proteins. COMMD1, the prototype of this family, has been associated with numerous diseases such as hepatic copper toxicity syndrome, hypercholesterolemia and cancer, but the biological role of the other nine members remains largely unknown. Our study show for the first time that in the liver COMMD6 and COMMD9 both an important role have to preserve cholesterol and copper homeostasis, similarly as we previously demonstrated for COMMD1. Interestingly, however, our data indicate that only myeloid COMMD1 prevents uncontrolled inflammation but not COMMD6 and COMMD9. We show that the COMMD proteins form together a stable multi-COMMD protein complex to regulate these cellular processes. The organization of this complex is likely cell type-specific, but the exact composition of these complexes remains unclear, and more research is wanted. Taken together our work revealed that the COMMD proteins likely act together to facilitate the endosomal trafficking of different transmembrane proteins such as LDLR and ATP7B to preserve cholesterol and copper homeostasis. We expect that better understanding of these pathways will advance therapeutic research to treat hypercholesterolemia and copper disorders

Functional understanding of the versatile protein copper metabolism MURR1 domain 1 (COMMD1) in copper homeostasis

Copper is an important cofactor in numerous biological processes in all living organisms. However, excessive copper can be extremely toxic, so it is vital that the copper level within a cell is tightly regulated. The damaging effect of copper is seen in several hereditary forms of copper toxicity in humans and animals. At present, Wilson's disease is the best-described and best-studied copper-storage disorder in humans; it is caused by mutations in the ATP7B gene. In dogs, a mutation in the COMMD1 gene has been found to be associated with copper toxicosis. Using a liver-specific Commd1 knockout mouse, the biological role of Commd1 in copper homeostasis has been confirmed. Yet, the exact mechanism by which COMMD1 regulates copper homeostasis is still unknown. Here, we give an overview of the current knowledge and perspectives on the molecular function of COMMD1 in copper homeostasis

Endosomal receptor trafficking: Retromer and beyond

The tubular endolysosomal network is a quality control system that ensures the proper delivery of internalized receptors to specific subcellular destinations in order to maintain cellular homeostasis. Although retromer was originally described in yeast as a regulator of endosome-to-Golgi receptor recycling, mammalian retromer has emerged as a central player in endosome-to-plasma membrane recycling of a variety of receptors. Over the past decade, information regarding the mechanism by which retromer facilitates receptor trafficking has emerged, as has the identification of numerous retromer-associated molecules including the WASH complex, sorting nexins and TBC1d5. Moreover, the recent demonstration that several sorting nexins can directly interact with retromer cargo to facilitate endosome-to-Golgi retrieval has provided new insight into how these receptors are trafficked in cells. The mechanism by which sorting nexin 17 cargoes are recycled out of the endosomal system was demonstrated to involve a retromer-like complex termed the retriever, which is recruited to WASH positive endosomes through an interaction with the COMMD/CCDC22/CCDC93 (CCC) complex. Lastly, the mechanisms by which bacterial and viral pathogens highjack this complex sorting machinery in order to escape the endolysosomal system or remain hidden within the cells are beginning to emerge. In this review, we will highlight recent studies that have begun to unravel the intricacies by which the retromer and associated molecules contribute to receptor trafficking and how deregulation at this sorting domain can contribute to disease or facilitate pathogen infection.This is the peer reviewed version of the following article: Wang, Jing, Alina Fedoseienko, Baoyu Chen, Ezra Burstein, Da Jia, and Daniel D. Billadeau. "Endosomal receptor trafficking: Retromer and beyond." Traffic 19, no. 8 (2018): 578-590, which has been published in final form at doi:10.1111/tra.12574. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited

A cell-type-specific role for murine Commd1 in liver inflammation

The transcription factor NF-κB plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-κB activation may protect tissues from stress, a prolonged NF-κB activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-κB activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-κB transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD

Antibody validation and exploratory immunostaining for COMMD1 in human ovarian tumor samples.

<p><b>(A)</b> Representative immunohistochemical COMMD1 staining in paraffin embedded HEK293T and HeLa cells depleted for COMMD1. <b>(B)</b> HEK293T and HeLa cells were stably silenced for COMMD1 as shown by immunoblotting. (<b>C</b>) Observational immunostainings for COMMD1, including its control IgG₁ immunostaining in a consecutive slide, in HGSOC patient samples demonstrating either absent or presence of nuclear COMMD1. (<b>D</b>) Quantification workflow of immunohistochemical COMMD1 staining. Image analysis was performed using the ImageJ-based software package FIJI. DAB staining and hematoxylin staining were deconvoluted and images were subsequently converted into 8-bit gray scale images. Hematoxylin staining was used to define cytoplasm/nucleus boundaries. Vectors were subsequently used to measure DAB staining intensities across cells and quantify nuclear COMMD1 levels in relation to cytoplasmic levels. (B) Three ‘nuclear COMMD1-negative’ (n = 10 cells per tumor sample) and three ‘nuclear COMMD1-positive’ tumor samples were analyzed. Averages and standard deviations are indicated. Relative nuclear COMMD1 levels to cytoplasmic levels are plotted per tumor sample.</p