The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.00

Functional phosphatome requirement for protein homeostasis, networked mitochondria, and sarcomere structure in C. elegans muscle

Background: Skeletal muscle is central to locomotion and metabolic homeostasis. The laboratory worm C. elegans has been developed into a genomic model for assessing the genes and signals that regulate muscle development and protein degradation. Past work has identified a receptor tyrosine kinase signalling network that combinatorially controls autophagy, nerve signal to muscle to oppose proteasome based degradation, and extracellular matrix based signals that control calpain and caspase activation. The last two discoveries were enabled by following up results from a functional genomic screen of known regulators of muscle. Recently, a screen of the kinome requirement for muscle homeostasis identified roughly 40% of kinases as required for C. elegans muscle health; 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle. To complement this kinome screen, here we screen most of the phosphatases in C. elegans. Methods: RNAi was used to knockdown phosphatase encoding genes. Knockdown was first conducted during development with positive results also knocked down only in fully developed adult muscle. Protein homeostasis, mitochondrial structure, and sarcomere structure were assessed using transgenic reporter proteins. Genes identified as being required to prevent protein degradation were also knocked down in conditions that blocked proteasome or autophagic degradation. Genes identified as being required to prevent autophagic degradation were also assessed for autophagic vesicle accumulation using another transgenic reporter. Lastly, bioinformatics were used to look for overlap between kinases and phosphatases required for muscle homeostasis and the prediction that one phosphatase was required to prevent MAPK activation was assessed by Western blot. Results: A little over half of all phosphatases are each required to prevent abnormal development or maintenance of muscle. 86 of these phosphatase have known human orthologues, 57 of which are known to be expressed in human skeletal muscle. Of the phosphatases required to prevent abnormal muscle protein degradation, roughly half are required to prevent increased autophagy. Conclusions: A significant portion of both the kinome and phosphatome are required for establishing and maintaining C. elegans muscle health. Autophagy appears to be the mostly commonly triggered form of protein degradation in response to disruption of phosphorylation based signalling. The results from these screens provide measurable phenotypes for analysing the combined contribution of kinases and phosphatases in a multi-cellular organism and suggest new potential regulators of human skeletal muscle for further analysis

The identification of a novel role for BRCA1 in regulating RNA Polymerase I transcription

The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA polymerase I (Pol-I) and its associated holoenzyme complex. Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene. We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened. We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage

Alteration of AKT Activity Increases Chemotherapeutic Drug and Hormonal Resistance in Breast Cancer yet Confers an Achilles Heel by Sensitization to Targeted Therapy

The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/ MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance. Orignally published Advances in Enzyme Regulation, Vol. 48, No. 1, 2008

Phase I/II study of verteporfin photodynamic therapy in locally advanced pancreatic cancer

Background:Patients with pancreatic cancer have a poor prognosis apart from the few suitable for surgery. Photodynamic therapy (PDT) produces localised tissue necrosis but previous studies using the photosensitiser meso-tetrahydroxyphenylchlorin (mTHPC) caused prolonged skin photosensitivity. This study assessed a shorter acting photosensitiser, verteporfin.Methods: Fifteen inoperable patients with locally advanced cancers were sensitised with 0.4 mg kg-1 verteporfin. After 60-90 min, laser light (690 nm) was delivered via single (13 patients) or multiple (2 patients) fibres positioned percutaneously under computed tomography (CT) guidance, the light dose escalating (initially 5 J, doubling after each three patients) until 12 mm of necrosis was achieved consistently.Results:In all, 12 mm lesions were seen consistently at 40 J, but with considerable variation in necrosis volume (mean volume 3.5 cm 3 at 40 J). Minor, self-limiting extrapancreatic effects were seen in multifibre patients. No adverse interactions were seen in patients given chemotherapy or radiotherapy before or after PDT. After PDT, one patient underwent an R0 Whipple's pancreaticoduodenectomy.Conclusions:Verteporfin PDT-induced tumour necrosis in locally advanced pancreatic cancer is feasible and safe. It can be delivered with a much shorter drug light interval and with less photosensitivity than with older compounds. © 2014 Cancer Research UK

Age-Related Disparities in Trauma Center Access for Severe Head Injuries Following the Release of the Updated Field Triage Guidelines

Objective: In 2006, the American College of Surgeons’ Committee on Trauma and the Center for Disease Control released field triage guidelines with special consideration for older adults. Additional considerations for direct transport to a Level I or II trauma center (TC) were added in 2011, reflecting perceived undertriage to TCs for older adults. We examined whether age-based disparities in TC care for severe head injury decreased following introduction of the 2011 revisions. Methods: A pre-post design analyzing the 2009 and 2012 Healthcare Cost and Utilization Project State Emergency Department Databases (SEDD) and State Inpatient Databases (SID) with multivariable logistic regressions considered changes in (1) the trauma designation of the emergency department where treatment was initiated and (2) transfer to a TC following initial treatment at a non-TC. Results: Compared with adults aged 18–44 years, after multivariable adjustment, in both years TC care was less likely for adults aged 45–64 years (OR: 0.76 in 2009 and 0.74 in 2012), aged 65–84 years (OR: 0.61 and 0.59), and aged 85+ years (OR: 0.53 and 0.56). Between 2009 and 2012, the likelihood of TC care increased for all age groups, with the largest increase among those aged 85+ years (OR = 1.18), which was statistically different (p = .02) from the increase among adults aged 18–44 years (OR = 1.12). The analysis of transfers yielded similar results. Conclusions: Although patterns of increased TC treatment for all groups with severe head trauma indicate improvements, age-based disparities persisted

Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative (BPD)

Benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) is currently under investigation as a photosensitizer for photodynamic therapy (PDT). Since BPD exhibits rapid pharmacokinetics in plasma and tissues, we assessed damage to tumour and muscle microvasculature when light treatment for PDT was given at short times after injection of photosensitizer. Groups of rats with chondrosarcoma were given 2 mg kg−1 of BPD intravenously 5 min to 180 min before light treatment of 150 J cm−2 690 nm. Vascular response was monitored using intravital microscopy and tumour cure was monitored by following regrowth over 42 days. For treatment at 5 or 30 min after BPD injection, blood flow stasis was limited to tumour microvasculature with lesser response in the surrounding normal microvasculature, indicating selective targeting for damage. No acute changes were observed in vessels when light was given 180 min after BPD injection. Tumour regression after light treatment occurred in all animals given PDT with BPD. Long-term tumour regression was greater in animals treated 5 min after BPD injection and least in animals given treatment 180 min after drug injection. The correlation between the timing for vascular damage and cure implies that blood flow stasis plays a significant role in PDT-induced tumour destruction. © 1999 Cancer Research Campaig

Genetic Screening to Find Novel Regulators of Tumor Suppressor Homolog Kinase Responsive to Stress B (krsB)

Biological Science

Theory and Practice of Business Process Management

Traditional model of management is based on hierarchical decomposition of organizational structure. Company is divided on workroom, union, partitions and every formation of his has independent agenda and his responsibility. However the formations often have tendency create about themselves barrier, especially communications and informatics barrier. Compared to that, process management is relatively new view of organization that moves activities of many companies. Process organization tries organizing and managing the work like comprehensive complex, which is of further distributed on individual sub-processes, which are logically linked. It is known; that the BPM is exploited in many line productions, nonproductive and tertiary sphere and his conventions pays in the same way in all lines. This statement I can corroborate thanks to my research results (below). Within the overall context of this research, we have understood the aspects of BPM to mean: a view and standpoint on the problems and issues related to the management of enterprises` processes and this includes such areas as aims, factors, components, support as well as the benefits of BPM itself. As it was indicated in the title of this paper, we mainly concentrate here on presenting the main results of the last-mentioned research area, i.e. where we evaluate the benefits of PM from the managements of Czech enterprises` point-of-view. The results are shown as a summary of all of the companies (respondents), of which there were 132