The Copper Metabolism MURR1 Domain containing 1 protein COMMD1 has been associated with copper homeostasis, NF-kappaB signaling, and sodium transport. Recently, we identified COMMD1 as a novel protein in HIF-1 signaling. Mouse embryos deficient for Commd1 have increased expression of hypoxia/HIF-regulated genes i.e. VEGF, PGK and Bnip3. Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis, which control angiogenesis, erythropoiesis, glycolysis and cell survival/proliferation under normal and pathologic conditions. Although HIF activity is mainly controlled by ubiquitination and protein degradation by the von Hippel Lindau (pVHL) tumor suppressor gene other mechanisms have recently been identified that regulate HIF signaling independently of pVHL.

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

BubR1 is a key component of the spindle assembly checkpoint (SAC). Mutations that reduce BubR1 abundance cause aneuploidization and tumorigenesis in humans and mice, whereas BubR1 overexpression protects against these. However, how supranormal BubR1 expression exerts these beneficial physiological impacts is poorly understood. Here, we used Bub1b mutant transgenic mice to explore the role of the amino-terminal (BubR1(N)) and internal (BubR1(I)) Cdc20-binding domains of BubR1 in preventing aneuploidy and safeguarding against cancer. BubR1(N) was necessary, but not sufficient to protect against aneuploidy and cancer. In contrast, BubR1 lacking the internal Cdc20-binding domain provided protection against both, which coincided with improved microtubule-kinetochore attachment error correction and SAC activity. Maximal SAC reinforcement occurred when both the Phe- and D-box of BubR1(I) were disrupted. Thus, while under- or overexpression of most mitotic regulators impairs chromosome segregation fidelity, certain manipulations of BubR1 can positively impact this process and therefore be therapeutically exploited.

STAP1, encoding for STAP1 (signal transducing adaptor family member 1), has been reported as a candidate gene associated with familial hypercholesterolemia. Unlike established familial hypercholesterolemia genes, expression of STAP1 is absent in liver but mainly observed in immune cells. In this study, we set out to validate STAP1 as a familial hypercholesterolemia gene. Approach and Results: A whole-body Stap1 knockout mouse model (Stap1-/-) was generated and characterized, without showing changes in plasma lipid levels compared with controls. In follow-up studies, bone marrow from Stap1-/- mice was transplanted to Ldlr-/- mice, which did not show significant changes in plasma lipid levels or atherosclerotic lesions. To functionally assess whether STAP1 expression in B cells can affect hepatic function, HepG2 cells were cocultured with peripheral blood mononuclear cells isolated from heterozygotes carriers of STAP1 variants and controls. The peripheral blood mononuclear cells from STAP1 variant carriers and controls showed similar LDLR mRNA and protein levels. Also, LDL (low-density lipoprotein) uptake by HepG2 cells did not differ upon coculturing with peripheral blood mononuclear cells isolated from either STAP1 variant carriers or controls. In addition, plasma lipid profiles of 39 carriers and 71 family controls showed no differences in plasma LDL cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and lipoprotein(a) levels. Similarly, B-cell populations did not differ in a group of 10 STAP1 variant carriers and 10 age- and sex-matched controls. Furthermore, recent data from the UK Biobank do not show association between STAP1 rare gene variants and LDL cholesterol.

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of β-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.

Chronic inflammation is considered a causal risk factor predisposing to insulin resistance. However, evidence is accumulating that inflammation confined to the liver may not be causal to metabolic dysfunction. To investigate this, we assessed if hepatic inflammation explains the predisposition towards insulin resistance in low-density lipoprotein receptor knock-out (Ldlr (-/-)) mice. For this, wild type (WT) and Ldlr (-/-) mice were fed a chow diet, a high fat (HF) diet, or a high fat, high cholesterol (HFC) diet for 2 weeks. Plasma lipid levels were elevated in chow-fed Ldlr (-/-) mice compared to WT mice. Although short-term HF or HFC feeding did not result in body weight gain and adipose tissue inflammation, dyslipidemia was worsened in Ldlr (-/-) mice compared to WT mice. In addition, dyslipidemic HF-fed Ldlr (-/-) mice had a higher hepatic glucose production rate than HF-fed WT mice, while peripheral insulin resistance was unaffected. This suggests that HF-fed Ldlr (-/-) mice suffered from hepatic insulin resistance. While HFC-fed Ldlr (-/-) mice displayed the anticipated increased hepatic inflammation, this did neither exacerbate systemic nor hepatic insulin resistance. Therefore, our results show that hepatic insulin resistance is unrelated to cholesterol-induced hepatic inflammation in Ldlr (-/-) mice, indicating that hepatic inflammation may not contribute to metabolic dysfunction per se.

Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

A homozygous truncating frameshift mutation in CEP57 (CEP57T/T) has been identified in a subset of mosaic-variegated aneuploidy (MVA) patients; however, the physiological roles of the centrosome-associated protein CEP57 that contribute to disease are unknown. To investigate these, we have generated a mouse model mimicking this disease mutation. Cep57T/T mice died within 24 hours after birth with short, curly tails and severely impaired vertebral ossification. Osteoblasts in lumbosacral vertebrae of Cep57T/T mice were deficient for Fgf2, a Cep57 binding partner implicated in diverse biological processes, including bone formation. Furthermore, a broad spectrum of tissues of Cep57T/T mice had severe aneuploidy at birth, consistent with the MVA patient phenotype. Cep57T/T mouse embryonic fibroblasts and patient-derived skin fibroblasts failed to undergo centrosome maturation in G2 phase, causing premature centriole disjunction, centrosome amplification, aberrant spindle formation, and high rates of chromosome missegregation. Mice heterozygous for the truncating frameshift mutation or a Cep57-null allele were overtly indistinguishable from WT mice despite reduced Cep57 protein levels, yet prone to aneuploidization and cancer, with tumors lacking evidence for loss of heterozygosity. This study identifies Cep57 as a haploinsufficient tumor suppressor with biologically diverse roles in centrosome maturation and Fgf2-mediated bone formation.

Cells respond to cytotoxic DNA double-strand breaks (DSBs) by recruiting DNA repair proteins to the damaged site. This recruitment is dependent on ubiquitylation of adjacent chromatin areas by E3 ubiquitin ligases such as RNF8 and RNF168, which are recruited sequentially to the DSBs. However, it is unclear what dictates the sequential order and recruits RNF168 to the DNA lesion. Here, we reveal that L3MBTL2 (lethal(3)malignant brain tumour-like protein 2) is the missing link between RNF8 and RNF168. We found that L3MBTL2 is recruited by MDC1 and subsequently ubiquitylated by RNF8. Ubiquitylated L3MBTL2, in turn, facilitates recruitment of RNF168 to the DNA lesion and promotes DNA DSB repair. These results identify L3MBTL2 as a key target of RNF8 following DNA damage and demonstrates how the DNA damage response pathway is orchestrated by ubiquitin signalling.

The CCNE1 locus, which encodes cyclin E1, is amplified in many types of cancer cells and is activated in hepatocellular carcinomas (HCCs) from patients infected with hepatitis B virus or adeno-associated virus type 2, due to integration of the virus nearby. We investigated cell-cycle and oncogenic effects of cyclin E1 overexpression in tissues of mice.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Atherosclerosis is a chronic inflammatory disease driven by hypercholesterolemia. During aging, T cells accumulate cholesterol, potentially affecting inflammation. However, the effect of cholesterol efflux pathways mediated by ATP-binding cassette A1 and G1 (ABCA1/ABCG1) on T cell-dependent age-related inflammation and atherosclerosis remains poorly understood. In this study, we generate mice with T cell-specific Abca1/Abcg1-deficiency on the low-density-lipoprotein-receptor deficient (Ldlr-/-) background. T cell Abca1/Abcg1-deficiency decreases blood, lymph node, and splenic T cells, and increases T cell activation and apoptosis. T cell Abca1/Abcg1-deficiency induces a premature T cell aging phenotype in middle-aged (12-13 months) Ldlr-/- mice, reflected by upregulation of senescence markers. Despite T cell senescence and enhanced T cell activation, T cell Abca1/Abcg1-deficiency decreases atherosclerosis and aortic inflammation in middle-aged Ldlr-/- mice, accompanied by decreased T cells in atherosclerotic plaques. We attribute these effects to T cell apoptosis downstream of T cell activation, compromising T cell functionality. Collectively, we show that T cell cholesterol efflux pathways suppress T cell apoptosis and senescence, and induce atherosclerosis in middle-aged Ldlr-/- mice.

Hepatic lipid accumulation and mitochondrial dysfunction are hallmarks of metabolic associated fatty liver disease (MAFLD), yet molecular parameters underlying MAFLD progression are not well understood. Differential methylation within the mitochondrial DNA (mtDNA) has been suggested to be associated with dysfunctional mitochondria, also during progression to Metabolic Steatohepatitis (MeSH). This study further investigates whether mtDNA methylation is associated with hepatic lipid accumulation and MAFLD.

Obesity-related chronic inflammation plays an important role in the development of Metabolic Associated Fatty Liver Disease (MAFLD). Although the contribution of the pro-inflammatory NF-κB signaling pathway to the progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is well-established, its role as an initiator of hepatic steatosis and the underlying mechanism remains unclear. Here, we investigated the hypothesis that the hepatocytic NF-κB signaling pathway acts as a metabolic regulator, thereby promoting hepatic steatosis development.

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 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.

Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD-CCDC22 [coiled-coil domain containing 22]-CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma low-density lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear.