HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.     

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13–1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.Extracellular vesicles (EVs)¹ are membrane surrounded structures that exist in all body fluids and all cells studied so far release EVs (1). They are heterogeneous, spherical organelles spanning between 30 to more than 1000 nm in diameter and include exosomes, microvesicles, and apoptotic bodies (2). There is increasing evidence supporting the important role of EVs in cell-to-cell communication by their delivery of proteins, lipids, and nucleic acids from one donor cell to many target cells. The generation of exosomes/prostasomes is a complicated process involving two invagination sessions of biological membranes. The first one comprises the plasma membrane contributing with endocytic vesicles in the formation of early endosomes that mature into late endosomes. The second one starts multiple inward buddings of the late endosomal membrane creating intraluminal vesicles (ILVs) therewith completing formation of multivesicular bodies (MVBs) or storage vesicles (3) thus retaining selected molecules from the maternal cell. Ceramide can induce such formation of small microdomains into larger domains (4). Ceramide is one of two cleavage products of sphingomyelin by sphingomyelinase, the other is phosphocholine (5) and prostasomes contain sphingomyelinase (6). The membrane of MVBs (storage vesicles) may fuse with the plasma membrane of the secretory cell and, in case of prostate epithelial cells, release the intraluminal vesicles as prostasomes to the extracellular space (7, 8). It is noteworthy that the bilayered membrane surrounding prostasomes (after the two sessions of invaginations) should be regarded as “right-side-out” with reference to the plasma membrane. This is illustrated by e.g. Mg²⁺ and Ca²⁺ -stimulated ATPase that is an ectoenzyme (9) that is also appearing at the outer surface of prostasomes (10). The corollary is that cell surface interactive molecules like enzymes and receptors may appear also on the membranes of exosomes/prostasomes.The majority of prostasomes ranges in diameter-size from 30–200 nm, with a mean of 142 nm (11). The main purpose of prostasomes may be to transfer newly synthesized proteins from the prostate gland to sperm and thereby, among other things, render them protection in the female genital tract (12, 13). Prostasomal proteins may be transferred to sperm through different mechanisms, viz direct interaction with the sperm membrane (14), fusion at a lowered pH (15), and internalization (16). Prostasomes are immunosuppressive and regulate the complement system and they have proven antioxidant and antibacterial properties (17, 18). Prostasomes contain a surrounding lipid membrane bilayer that exhibits a high cholesterol/phospholipid ratio (19). The lipid composition of the membrane is unusual and among the phospholipids sphingomyelin is the dominant one, contrary to other cell membranes where phosphatidylcholine is most abundant. Prostasomes have a strong contribution of saturated and monounsaturated fatty acids (19, 20). These characteristics together with a high cholesterol/phospholipid ratio make the membrane of the prostasome very stable as demonstrated by electron spin resonance (19).In the early 1970s the plasma membrane of the cell was described as a fluid mosaic by Singer and Nicholson (21), but as early as in 1953 Palade claimed that in the bilayered lipid membrane, proposed by Davson and Danielli (22), were areas of different composition, so called caveolae (23). These caveolae are invaginations of the plasma membrane (24). The first hypothesis of lipid rafts (specialized membrane domains enriched in glycosphingolipids, proteins and cholesterol) was brought up in 1988 by van Meer and Simons (25) and was subsequently elaborated in 1997 by Simons and Ikonen (26). Lipid rafts were defined as low density subdomains of the plasma membrane that are resistant to nonionic detergents at a low temperature (27, 28). Fatty acids present in lipid rafts are more saturated, compared with the membrane adjacent to the domains. It means that the fatty acids can be packed more densely and this may lead to phase separation. The abundance of intercalating cholesterol makes the rafts more rigid and less fluid than the rest of the plasma membrane (29). In other words, the membrane can undergo phase separation into co-existing liquid-disordered and liquid-ordered phases. The liquid-ordered phase (the lipid raft) becomes enriched in cholesterol and saturated fatty acids and is characterized by tight lipid packing and reduced molecular diffusion, as we noticed for prostasomes (19).There are two different types of lipid rafts, planar and caveolae. The distinguishing factor is that the caveolae are formed by the protein caveolin whereas the planar rafts lack this protein (30). Instead they contain the protein flotillin (31). Researchers have found that selected proteins localize, and colocalize in lipid rafts (32). Lipid rafts are not anchored at a specific site in the plasma membrane, but float freely. This enables larger and more stable platform domains to aggregate (33). The formed aggregates are involved in many biological functions including endocytosis, cell communication, molecular trafficking, neurotransmission and they could be understood as organizing centers for signaling molecules and receptors (30, 31). When cells are depleted of cholesterol, e.g. by the agent methyl-β-cyclodextrin, formation of caveolae expression and also raft-dependent endocytosis are inhibited (34). This demonstrates the importance of these cholesterol-enriched domains to cell survival. Flotillins are also involved in endocytosis in a process controlled by the phosphorylation of tyrosine residues (35).In this work we asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Accordingly, we prepared lipid rafts from human prostasomes in order to characterize their protein content.     

Methylomic analysis of monozygotic twins discordant for childhood psychotic symptoms

Childhood psychotic symptoms are associated with increased rates of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood; thus, elucidating early risk indicators is crucial to target prevention efforts. There is considerable discordance for psychotic symptoms between monozygotic twins, indicating that child-specific non-genetic factors must be involved. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to childhood psychotic symptoms. Therefore, this study explored whether differences in DNA methylation at age 10 were associated with monozygotic twin discordance for psychotic symptoms at age 12. The Environmental Risk (E-Risk) Longitudinal Twin Study cohort of 2,232 children (1,116 twin pairs) was assessed for age-12 psychotic symptoms and 24 monozygotic twin pairs discordant for symptoms were identified for methylomic comparison. Children provided buccal samples at ages 5 and 10. DNA was bisulfite modified and DNA methylation was quantified using the Infinium HumanMethylation450 array. Differentially methylated positions (DMPs) associated with psychotic symptoms were subsequently tested in post-mortem prefrontal cortex tissue from adult schizophrenia patients and age-matched controls. Site-specific DNA methylation differences were observed at age 10 between monozygotic twins discordant for age-12 psychotic symptoms. Similar DMPs were not found at age 5. The top-ranked psychosis-associated DMP (cg23933044), located in the promoter of the C5ORF42 gene, was also hypomethylated in post-mortem prefrontal cortex brain tissue from schizophrenia patients compared to unaffected controls. These data tentatively suggest that epigenetic variation in peripheral tissue is associated with childhood psychotic symptoms and may indicate susceptibility to schizophrenia and other mental health problems.     

Sex differences in infant mortality in Spitalfields, London, 1750-1839

This study examines sex differences in infant mortality in Spitalfields, London, and the estimated contribution of endogenous and exogenous factors to neonatal and infant mortality using the biometric model from 1750 to 1839. There was a marked decline in the risk of death during infancy and the neonatal period for both sexes during the study period. There was significant excess male infant mortality compared with that of females in the 1750-59 cohort, estimated from baptism and burial registers, but not in later cohorts. Similarly, males had higher neonatal mortality rates than females in 1750-59 but not in later cohorts. Biometric analyses suggest that the observed decrease in neonatal mortality in both sexes was caused by a reduction in both endogenous and exogenous causes of death. The contribution of maternal health and breast-feeding practices to the observed patterns of mortality is discussed in the light of available evidence.     

Evaluation of Phosphatidylinositol-4-Kinase IIIα as a Hepatitis C Virus Drug Target

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.     

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific (15)N relaxation and (1)H-(15)N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.