Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid–based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth

Computed tomographic analysis of the quality of trunk muscles in asymptomatic and symptomatic lumbar discectomy patients

Background: No consensus exists on how rehabilitation programs for lumbar discectomy patients with persistent complaints after surgery should be composed. A better understanding of normal and abnormal postoperative trunk muscle condition might help direct the treatment goals. Methods: A three-dimensional CT scan of the lumbar spine was obtained in 18 symptomatic and 18 asymptomatic patients who had undergone a lumbar discectomy 42 months to 83 months (median 63 months) previously. The psoas muscle (PS), the paraspinal muscle mass (PA) and the multifidus muscle (MF) were outlined at the L3, L4 and L5 level. Of these muscles, fat free Cross Sectional Area (CSA) and fat CSA were determined. CSA of the lumbar erector spinae (LES = longissimus thoracis + iliocostalis lumborum) was calculated by subtracting MF CSA from PA CSA. Mean muscle CSA of the left and right sides was calculated at each level. To normalize the data for interpersonal comparison, the mean CSA was divided by the CSA of the L3 vertebral body (mCSA = normalized fat-free muscle CSA; fCSA = normalized fat CSA). Differences in CSA between the pain group and the pain free group were examined using a General Linear Model (GLM). Three levels were examined to investigate the possible role of the level of operation. Results: In lumbar discectomy patients with pain, the mCSA of the MF was significantly smaller than in pain-free subjects (p = 0.009) independently of the level. The mCSA of the LES was significantly smaller in pain patients, but only on the L3 slice (p = 0.018). No significant difference in mCSA of the PS was found between pain patients and pain-free patients (p = 0.462). The fCSA of the MF (p = 0.186) and of the LES (p = 0.256) were not significantly different between both populations. However, the fCSA of the PS was significantly larger in pain patients than in pain-free patients. (p = 0.012). The level of operation was never a significant factor. Conclusions: CT comparison of MF, LES and PS muscle condition between lumbar discectomy patients without pain and patients with protracted postoperative pain showed a smaller fat-free muscle CSA of the MF at all levels examined, a smaller fat-free muscle CSA of the LES at the L3 level, and more fat in the PS in patients with pain. The level of operation was not found to be of importance. The present results suggest a general lumbar muscle dysfunction in the pain group, in particular of the deep stabilizing muscle system

Reoperations after first lumbar disc herniation surgery; a special interest on residives during a 5-year follow-up

BACKGROUND: The overall rate of operations after recurrent lumbar disc herniation has been shown to be 3–11%. However, little is known about the rate of residives. Thus the aim of this study was to explore the cumulative rates of re-operations and especially residive disc herniations at the same side and level as the primary disc herniation after first lumbar disc herniation surgery and the factors that influence the risk of re-operations over a five year follow-up study. METHODS: 166 virgin lumbar disc herniation patients (mean age 42 years, 57% males) were studied. Data on patients' initial disc operations and type and timing of re-operations during the follow-up were collected from patient files. Back and leg pain on visual analog scale and employment status were collected by questionnaires. RESULTS: The cumulative rate of re-operations for lumbar disc herniation was 10.2% (95% Cl 6.0 to 15.1). The rate of residives at initial site was 7.4% (95% Cl 3.7 to 11.3) and rate of lumbar disc herniations at other sites was 3.1% (95% Cl 0.6 to 6.2). The occurrence of residive lumbar disc herniations was evenly distributed across the 5 years. Neither age, gender, preoperative symptoms, physical activity nor employment had effect on the probability of re-operation. CONCLUSION: Seven percent of the lumbar disc patients had a residive lumbar disc operation within five years of their first operation. No specific factors influencing the risk for re-operation were found

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general

Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation

Arabidopsis thaliana phosphatidylinositol 3-kinase (AtVPS34) functions in the development and germination of pollen by catalyzing the biosynthesis of phosphatidylinositol 3-phosphate (PI3P). In yeast, Vps15p is required for the membrane targeting and activity of Vps34. The expression of Arabidopsis thaliana VPS15 (AtVPS15), an ortholog of yeast Vps15, is mainly detected in pollen grains and pollen tubes. To determine its role in pollen development and pollen tube growth, we attempted to isolate the T-DNA insertion mutants of AtVPS15; however, homozygous lines of atvps15 were not obtained from the progeny of atvps15/+ heterozygotes. Genetic analysis revealed that the abnormal segregation is due to the failure of transmission of the atvps15 allele through pollen. Most pollen grains from the atvps15/+ genotype are viable, with normal exine structure and nuclei, but some mature pollen grains are characterized with unusual large vacuoles that are not observed in pollen grains from the wild AtVPS15 genotype. The germination ratio of pollen from the atvps15/+ genotype is about half when compared to that from the wild AtVPS15 genotype. When supplied with PI3P, in vitro pollen germination of the atvps15/+ genotype is greatly improved. Presumably, AtVPS15 functions in pollen development and germination by regulating PI3P biosynthesis in Arabidopsis

The Homeodomain Protein Defective Proventriculus Is Essential for Male Accessory Gland Development to Enhance Fecundity in Drosophila

The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity

Control of Precursor Maturation and Disposal Is an Early Regulative Mechanism in the Normal Insulin Production of Pancreatic β-Cells

The essential folding and maturation process of proinsulin in β-cells is largely uncharacterized. To analyze this process, we improved approaches to immunoblotting, metabolic labeling, and data analysis used to determine the proportion of monomers and non-monomers and changes in composition of proinsulin in cells. We found the natural occurrence of a large proportion of proinsulin in various non-monomer states, i.e., aggregates, in normal mouse and human β-cells and a striking increase in the proportion of proinsulin non-monomers in Ins2+/Akita mice in response to a mutation (C96Y) in the insulin 2 (Ins2) gene. Proinsulin emerges in monomer and abundant dual-fate non-monomer states during nascent protein synthesis and shows heavy and preferential ATP/redox-sensitive disposal among secretory proteins during early post-translational processes. These findings support the preservation of proinsulin's aggregation-prone nature and low relative folding rate that permits the plentiful production of non-monomer forms with incomplete folding. Thus, in normal mouse/human β-cells, proinsulin's integrated maturation and degradation processes maintain a balance of natively and non-natively folded states, i.e., proinsulin homeostasis (PIHO). Further analysis discovered the high susceptibility of PIHO to cellular energy and calcium changes, endoplasmic reticulum (ER) and reductive/oxidative stress, and insults by thiol reagent and cytokine. These results expose a direct correlation between various extra-/intracellular influences and (a)typical integrations of proinsulin maturation and disposal processes. Overall, our findings demonstrated that the control of precursor maturation and disposal acts as an early regulative mechanism in normal insulin production, and its disorder is crucially linked to β-cell failure and diabetes pathogenesis

Autophagy fights disease through cellular self-digestion

Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62766/1/nature06639.pd

Genome-Wide Functional Profiling Identifies Genes and Processes Important for Zinc-Limited Growth of Saccharomyces cerevisiae

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency