Roger Meier

BACKGROUND: Reverse cholesterol transport by HDLs (high-density lipoproteins) is considered an antiatherogenic metabolic pathway. Hepatocytes are the main contributors to the efficacy of this pathway by the production of apoA-I (apolipoprotein A1) and its lipidation by ABCA1 (ATP-binding cassette transporter A1), selective uptake of cholesterol via SR-BI (scavenger receptor class B type 1), and uptake of entire HDL particles. The molecular determinants of the latter step are not well understood. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of HDL fluorescently labeled at its protein moiety into Huh-7 hepatocarcinoma cells. Top hit genes were validated by targeted in vitro experiments and the analysis of associations between their variants and HDL-C (HDL-cholesterol) levels in the databases of the Global Lipids Genetics Consortium and the UK Biobank, as well as inborn errors of metabolism and their respective mouse models. RESULTS: The knockdown of 128 genes significantly inhibited HDL uptake. Six of them encode components of the COPI (coat protein I) coatomer, namely, COPA , COPB1 , COPB2 , COPG1 , ARCN1 , and COPZ1 . Knocking down any of them decreased the uptake of both fluorescently labeled proteins and lipids of HDL, the cell surface abundance of SR-BI, and APOA1 expression and apoA-I secretion but increased the cell surface abundance of ABCA1. Common single-nucleotide polymorphisms of ARCN1 and COPB1 were associated with significantly higher HDL-C levels in the population, while rare COPA and COPG1 variants causing immunopathies were associated with rather lower levels of HDL-C in both affected patients and the corresponding genetically modified mice. CONCLUSIONS: In hepatocytes, the COPI coatomer regulates HDL holoparticle uptake, selective lipid uptake, apoA-I secretion, and ABCA1 expression, and thereby, it influences plasma levels of HDL-C.

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.

Background: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR . The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25 , are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25 , overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum . PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5) ₂ ) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium . Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve ^- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila . We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes. Importance: Cells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogen Legionella .