Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Autophagy also plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as eliminating intracellular pathogens. Thus, autophagy is generally thought of as a survival mechanism, although its deregulation has been linked to non-apoptotic cell death. Autophagy can be either non-selective or selective in the removal of specific organelles, ribosomes and protein aggregates, although the mechanisms regulating aspects of selective autophagy are not fully worked out. In addition to elimination of intracellular aggregates and damaged organelles, autophagy promotes cellular senescence and cell surface antigen presentation, protects against genome instability and prevents necrosis, giving it a key role in preventing diseases such as cancer, neurodegeneration, cardiomyopathy, diabetes, liver disease, autoimmune diseases and infections. This review summarizes the most up-to-date findings on how autophagy is executed and regulated at the molecular level and how its disruption can lead to disease. Keywords autophagy; apoptosis; stress; mechanisms; energy; disease; cancer; neurodegeneration; infection What is autophagy?The term 'autophagy', derived from the Greek meaning 'eating of self', was first coined by Christian de Duve over 40 years ago, and was largely based on the observed degradation of mitochondria and other intra-cellular structures within lysosomes of rat liver perfused with the pancreatic hormone, glucagon [1]. The mechanism of glucagon-induced autophagy in the liver is still not fully understood at the molecular level, other than that it requires cyclic AMP induced activation of protein kinase-A and is highly tissue-specific [2]. In recent years the scientific world has 'rediscovered' autophagy, with major contributions to our molecular understanding and appreciation of the physiological significance of this process coming from numerous laboratories [3][4][5][6]. Although the importance of autophagy is well recognized in mammalian systems, many of the mechanistic breakthroughs in delineating how autophagy is regulated and executed at the molecular level have been made in yeast (Saccharomyces cerevisiae) [3, Copyright © 2010 There are three defined types of autophagy: macro-autophagy, micro-autophagy, and chaperone-mediated autophagy, all of which promote proteolytic degradation of cytosolic components at the lysosome. Macro-autophagy delivers cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle, referred to as an autophagosome, that fuses with the lysosome to form an autolysosome. In micro-autophagy, by contrast, cytosolic components are directly taken up by the lysosome itself through invagination of the lysosomal membrane. Both macro-and micro-autophagy are able to engulf large structures throu...

Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure ‘autophagic flux’ has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.

We conducted a genome-wide association study for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in 401 affected individuals and 1,323 controls, with replication in an independent sample of 793 NSCL/P triads. We report two new loci associated with NSCL/P at 17q22 (rs227731, combined P = 1.07 × 10 −8 , relative risk in homozygotes = 1.84, 95% CI 1.34-2.53) and 10q25.3 (rs7078160, combined P = 1.92 × 10 −8 , relative risk in homozygotes = 2.17, 95% CI 1.32-3.56).NSCL/P is one of the most common human birth defects. In European populations, NSCL/P has a prevalence ranging from 1 in 700 to 1 in 1,000. We recently reported a susceptibility locus for NSCL/P at chromo some 8q24.21 from a genome wide association study in 224 individuals with NSCL/P (cases) and 383 population based controls 1 . This locus is the second susceptibility locus to have been unequivocally identified for NSCL/P to date, the first being the IRF6 locus 2 .To identify additional cleft susceptibility loci, we enlarged our sample by genotyping an additional set of 177 NSCL/P cases and adding the genotypes of 940 population based controls of central European origin. Genotyping was performed using Illumina BeadChips (Human610 Quad and HumanHap 550k).Following quality control (Supplementary Methods and Supplementary Fig. 1), association analysis of 521,288 SNPs having a minor allele frequency (MAF) of ≥1% in controls was performed in 399 cases and 1,318 controls.After excluding markers from the previously described 8q24.21 locus, 20 SNPs with P < 10 −5 remained. Five chromosomal loci (8q12.3, 10q25.3, 13q31.1, 15q13.3 and 17q22) were located within these 20 top SNPs, and the associations at these loci were further supported by at least three more SNPs with P < 10 −4 ( Supplementary Fig. 2 and Supplementary Table 1). Two additional regions were considered to be promising NSCL/P susceptibility loci (6p22.1, 11q14.2), as they contained at least four markers with P < 10 −4 .To replicate the genome wide association study (GWAS) findings, we selected the 20 top SNPs (P < 10 −5 ) as well as additional backup markers for each of the seven previously mentioned loci, resulting in two replication assays. We included additional SNPs with P < 10 −4 in the two replication assays, giving highest priority to SNPs with the lowest P values. Thus, a total of 56 markers were genotyped in a replication sample of 793 NSCL/P triads of European origin. Genotyping using matrix assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry (Sequenom Inc.) was successful for 45 markers (representing 32 different loci), which were then analyzed by the transmission disequilibrium test in 665 triads (128 triads were excluded after quality control, Supplementary Methods).Of the 45 SNPs successfully genotyped, 11 (representing six differ ent loci) showed P < 0.05 in the replication sample (Supplementary Table 2). Two of these SNPs remained significant after correction for multiple testing by a conservative Bonferroni procedure (17q22: rs227731, P corr ...

We conducted a genome-wide association study involving 224 cases and 383 controls of Central European origin to identify susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). A 640-kb region at chromosome 8q24.21 was found to contain multiple markers with highly significant evidence for association with the cleft phenotype, including three markers that reached genome-wide significance. The 640-kb cleft-associated region was saturated with 146 SNP markers and then analyzed in our entire NSCL/P sample of 462 unrelated cases and 954 controls. In the entire sample, the most significant SNP (rs987525) had a P value of 3.34 x 10(-24). The odds ratio was 2.57 (95% CI = 2.02-3.26) for the heterozygous genotype and 6.05 (95% CI = 3.88-9.43) for the homozygous genotype. The calculated population attributable risk for this marker is 0.41, suggesting that this study has identified a major susceptibility locus for NSCL/P.

We have conducted the first meta-analyses for nonsyndromic cleft lip with or without cleft palate (NSCL/P) using data from the two largest genome-wide association studies published to date. We confirmed associations with all previously identified loci and identified six additional susceptibility regions (1p36, 2p21, 3p11.1, 8q21.3, 13q31.1 and 15q22). Analysis of phenotypic variability identified the first specific genetic risk factor for NSCLP (nonsyndromic cleft lip plus palate) (rs8001641; PNSCLP = 6.51 × 10−11; homozygote relative risk = 2.41, 95% confidence interval (CI) 1.84–3.16).

IntroductionThe hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes regulating mammalian oxygen homeostasis. 1 Among others, the corresponding gene products are involved in erythropoiesis, iron metabolism, angiogenesis, control of blood flow, glucose uptake and glycolysis, pH regulation, and cell-cycle control. 2 HIF-1 is a ␣ 1 ␤ 1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements. 3 Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein (pVHL) targets the HIF-1␣ subunit for rapid ubiquitination and proteasomal degradation. 4 Binding of the pVHL tumor suppressor protein requires the modification of HIF-1␣ by prolyl-4-hydroxylation at prolines 402 and 564 of human HIF-1␣. [5][6][7][8] A family of 3 oxygen-and iron-dependent prolyl-4-hydroxylases called PHD1, PHD2, PHD3, or HPH3, HPH2, HPH1, respectively, has been shown to hydroxylate HIF␣. 9,10 A fourth member, called PH-4, regulates HIF-1␣ in overexpression conditions only. 11 Thus, limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 This unusual mechanism of protein regulation provides the basis for the very rapid HIF-1␣ response to hypoxia. 13 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF-1␣ by factor inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity following a further decrease in oxygen availability. 14,15 Among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which coordinately regulate iron metabolism. [16][17][18][19][20] Increased iron uptake, release from the liver, plasma transport, and uptake in the bone marrow are essential to sustain the erythropoietic function of erythropoietin, the prototype HIF-1 target. Ceruloplasmin is a multicopper plasma protein containing ferroxidase activity necessary for Fe 3ϩ saturation of transferrin. 21 Hereditary aceruloplasminemia in humans as well as targeted deletion of the ceruloplasmin gene (Cp) in mice results in iron metabolism disorders characterized by anemia, hepatic iron overload, and neurodegeneration, demonstrating a tight connection between copper and iron metabolism. [22][23][24][25][26] Iron deficiency has been known for more than a decade to induce erythropoietin gene expression and HIF-1␣ protein stabilization. 27 Nowadays, these results are most likely explained by inactivation of the iron-dependent protein hydroxylases PHD1 to 3 and FIH. 12 Iron deficiency also results in mRNA induction of ceruloplasmin by HIF-1-dependent promoter activation and subsequent transcriptional up-regulation of the Cp gene. 20 Materials and methods Cell lines and cell cultureAll cell lines were cultured in Dulbecco modified Eagle medium (high glucose) as described previously. 29 Oxygen partial pressures in the hypoxic workstation (InVivO 2 -400; Ruskinn Technology, Leeds, United Kingdom) or in the incubator (M...

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-␣ subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-␣ for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione Stransferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.