Regulation of mTORC1 by phosphatidic acid: mechanism and structural insight

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase with remarkable control over cellular status. As a master regulator, mTOR integrates a variety of intra- and extra-cellular signals in order to coordinate them with appropriate gene expression, protein synthesis, metabolism, cell migration, autophagy – the list goes on! mTOR signaling, with involvement in so many important cellular processes, can have detrimental physiological effects when dysregulated. Aberrant mTOR signaling is now known to contribute to a great number of the major pathologies we face today. Understanding how mTOR is normally regulated is, therefore, important for informing the development of effective and specific therapeutics for diseases like cancer and diabetes. Significant research efforts over the last two decades have informed our current understanding of the extensive mTOR signaling pathways, but many questions remain. In my dissertation work I have investigated longstanding mysteries of mTOR activation by mitogens and nutrients, with specific focus on the mechanism by which the lipid second messenger, phosphatidic acid, activates mTOR complex 1. The mammalian target of rapamycin complex 1 (mTORC1) is regulated, in part, by the endogenous inhibitor DEPTOR. However, the mechanism of DEPTOR regulation with regard to rapid mTORC1 activation remains unknown. In collaboration with Dr. Mee Sup Yoon, I discovered that DEPTOR is rapidly and temporarily dissociated from mTORC1 upon mitogenic stimulation. We demonstrated that this mitogen stimulated DEPTOR dissociation is blocked by inhibition or depletion of the mTORC1 regulator, phospholipase D (PLD), and is recapitulated with the addition of the PLD product phosphatidic acid (PA). Parallel mass spectrometry analysis independently identified DEPTOR as an mTOR binding partner dissociated by PA. Interestingly, I found that only PA species with unsaturated fatty acid chains, such as those produced by PLD, are capable of displacing DEPTOR and activating mTORC1, with high affinity for the FRB domain of mTOR. Our findings, detailed in Chapter II reveal a mechanism of acute mTORC1 regulation that was previously unidentified and provide a molecular explanation for the exquisite specificity of PA function. In light of PA’s essential role in mTORC1 activation, I found it striking that mTOR proteins containing one of several point mutations have been reported to remain catalytically active in conditions when amino acids, and therefore PA, are absent. The existence of such hyperactive mTOR prompted me to ask whether a point mutation can render mTOR independent of regulation by PA and, if so, by what mechanism? In Chapter III, I describe how by examining the activity of several mTOR proteins each carrying a unique point mutation known to be associated with human cancer, I discovered that individual point mutations can confer varying degrees of PA-independent mTORC1 activity. My finding that an L1460P mutation in mTOR's FAT domain, S2215Y mutation in the kinase N-lobe, and E2419K in the kinase C-lobe all confer some mTORC1 activity in the absence of PA suggests that it is possible for PA-independent mTORC1 activity to result by more than one mechanism. Having identified that the activity of S2215Y mTOR is especially independent of PA and noting the implications of S2215Y for mTORC1’s structure, I propose that control of catalytic cleft access is a major aspect of mTORC1 activation by PA. My investigation also produced striking evidence that mTOR autophosphorylation, which is significantly hyperactivated by the R2505P mutation, does not necessarily correlate with PA-mediated mTORC1 phosphorylation of canonical substrates. Taken together, the experiments detailed in this chapter provide insight into the mechanism of mTORC1 activation by PA and carry significant therapeutic implications. Lastly, in Appendix A, I document several of my preliminary investigations of localization-dependent regulation of mTOR signaling. I have observed that both mTOR and raptor have a nuclear presence and that the proteins can localize there independently of each other. Additionally, I present evidence that nuclear mTORC1 phosphorylates the transcriptional repressor, Maf1, in a manner independent of the canonical mTORC1 pathway. Finally, I report for the first time that unsaturated PA species appear to recruit mTORC2 to detergent-sensitive cellular regions that I believe may be mitochondria-associated membranes

FPGA-based multi-view stereo system with flexible measurement setup

In recent years, stereoscopic image processing algorithms have gained importance for a variety of applications. To capture larger measurement volumes, multiple stereo systems are combined into a multi-view stereo (MVS) system. To reduce the amount of data and the data rate, calculation steps close to the sensors are outsourced to Field Programmable Gate Arrays (FPGAs) as upstream computing units. The calculation steps include lens distortion correction, rectification and stereo matching. In this paper a FPGA-based MVS system with flexible camera arrangement and partly overlapping field of view is presented. The system consists of four FPGA-based passive stereoscopic systems (Xilinx Zynq-7000 7020 SoC, EV76C570 CMOS sensor) and a downstream processing unit (Zynq Ultrascale ZU9EG SoC). This synchronizes the sensor near processing modules and receives the disparity maps with corresponding left camera image via HDMI. The subsequent computing unit calculates a coherent 3D point cloud. Our developed FPGA-based 3D measurement system captures a large measurement volume at 24 fps by combining a multiple view with eight cameras (using Semi-Global Matching for an image size of 640 px × 460 px, up to 256 px disparity range and with aggregated costs over 4 directions). The capabilities and limitation of the system are shown by an application example with optical non-cooperative surface

aLFQ: an R-package for estimating absolute protein quantities from label-free LC-MS/MS proteomics data

Motivation: The determination of absolute quantities of proteins in biological samples is necessary for multiple types of scientific inquiry. While relative quantification has been commonly used in proteomics, few proteomic datasets measuring absolute protein quantities have been reported to date. Various technologies have been applied using different types of input data, e.g. ion intensities or spectral counts, as well as different absolute normalization strategies. To date, a user-friendly and transparent software supporting large-scale absolute protein quantification has been lacking. Results: We present a bioinformatics tool, termed aLFQ, which supports the commonly used absolute label-free protein abundance estimation methods (TopN, iBAQ, APEX, NSAF and SCAMPI) for LC-MS/MS proteomics data, together with validation algorithms enabling automated data analysis and error estimation. Availability and implementation: aLFQ is written in R and freely available under the GPLv3 from CRAN (http://www.cran.r-project.org). Instructions and example data are provided in the R-package. The raw data can be obtained from the PeptideAtlas raw data repository (PASS00321). Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Intrathecal heat shock protein 60 mediates neurodegeneration and demyelination in the CNS through a TLR4- and MyD88-dependent pathway

Background Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo. Results We show here that HSP60 injected intrathecally causes neuronal and oligodendrocyte injury in the CNS in vivo through TLR4-dependent signaling. Intrathecal HSP60 results in neuronal cell death, axonal injury, loss of oligodendrocytes, and demyelination in the cerebral cortex of wild-type mice. In contrast both mice lacking TLR4 and the TLR adaptor molecule MyD88 are protected against deleterious effects induced by HSP60. In contrast to the exogenous TLR4 ligand, lipopolysaccharide, intrathecal HSP60 does not induce such a considerable inflammatory response in the brain. In the CNS, endogenous HSP60 is predominantly expressed in neurons and released during brain injury, since the cerebrospinal fluid (CSF) from animals of a mouse stroke model contains elevated levels of this stress protein compared to the CSF of sham- operated mice. Conclusions Our data show a direct toxic effect of HSP60 towards neurons and oligodendrocytes in the CNS. The fact that these harmful effects involve TLR4 and MyD88 confirms a molecular pathway mediated by the release of endogenous TLR ligands from injured CNS cells common to many forms of brain diseases that bi-directionally links CNS injury and activation of the innate immune system to neurodegeneration and demyelination in vivo

Efficacy of a brief manualized intervention Managing Cancer and Living Meaningfully (CALM) adapted to German cancer care settings: study protocol for a randomized controlled trial

Background: Although psycho-oncological interventions have been shown to significantly reduce symptoms of anxiety and depression and enhance quality of life, a substantial number of patients with advanced cancer do not receive psycho-oncological interventions tailored to their individual situation. Given the lack of reliable data on the efficacy of psycho-oncological interventions in palliative care settings, we aim to examine the efficacy of a brief, manualized individual psychotherapy for patients with advanced cancer: Managing Cancer and Living Meaningfully (CALM). CALM aims to reduce depression and death anxiety, to strengthen communication with health care providers, and to enhance hope and meaning in life. We adapted the intervention for German cancer care settings. Methods/Design: We use a single-blinded randomized-controlled trial design with two treatment conditions: intervention group (IG, CALM) and control group (CG). Patients in the CG receive a usual non-manualized supportive psycho-oncological intervention (SPI). Patients are randomized between the IG and CG and assessed at baseline (t0), after three (t1) and after 6 months (t2). We include patients with a malignant solid tumor who have tumor stages of III or IV (UICC classification). Patients who are included in the study are at least 18 years old, speak German fluently, score greater than or equal to nine on the PHQ-9 or/and greater than or equal to five on the Distress Thermometer. It is further necessary that there is no evidence of severe cognitive impairments. We measure depression, anxiety, distress, quality of life, demoralization, symptom distress, fatigue as well as spiritual well-being, posttraumatic growth and close relationship experiences using validated questionnaires. We hypothesize that patients in the IG will show a significantly lower level of depression 6 months after baseline compared to patients in the CG. We further hypothesize a significant reduction in anxiety and fatigue as well as significant improvements in psychological and spiritual well-being, meaning and post-traumatic growth in the IG compared to CG 6 months after baseline. Discussion: Our study will contribute important statistical evidence on whether CALM can reduce depression and existential distress in a German sample of advanced and highly distressed cancer patients

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms

The impact of single and pairwise Toll-like receptor activation on neuroinflammation and neurodegeneration

Background Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury. Methods Selected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry. Results Simultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo. Conclusions Sole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases’ diversity

Understanding the relation between Zika virus infection during pregnancy and adverse fetal, infant and child outcomes: a protocol for a systematic review and individual participant data meta-analysis of longitudinal studies of pregnant women and their infants and children

IntroductionZika virus (ZIKV) infection during pregnancy is a known cause of microcephaly and other congenital and developmental anomalies. In the absence of a ZIKV vaccine or prophylactics, principal investigators (PIs) and international leaders in ZIKV research have formed the ZIKV Individual Participant Data (IPD) Consortium to identify, collect and synthesise IPD from longitudinal studies of pregnant women that measure ZIKV infection during pregnancy and fetal, infant or child outcomes.Methods and analysisWe will identify eligible studies through the ZIKV IPD Consortium membership and a systematic review and invite study PIs to participate in the IPD meta-analysis (IPD-MA). We will use the combined dataset to estimate the relative and absolute risk of congenital Zika syndrome (CZS), including microcephaly and late symptomatic congenital infections; identify and explore sources of heterogeneity in those estimates and develop and validate a risk prediction model to identify the pregnancies at the highest risk of CZS or adverse developmental outcomes. The variable accuracy of diagnostic assays and differences in exposure and outcome definitions means that included studies will have a higher level of systematic variability, a component of measurement error, than an IPD-MA of studies of an established pathogen. We will use expert testimony, existing internal and external diagnostic accuracy validation studies and laboratory external quality assessments to inform the distribution of measurement error in our models. We will apply both Bayesian and frequentist methods to directly account for these and other sources of uncertainty.Ethics and disseminationThe IPD-MA was deemed exempt from ethical review. We will convene a group of patient advocates to evaluate the ethical implications and utility of the risk stratification tool. Findings from these analyses will be shared via national and international conferences and through publication in open access, peer-reviewed journals.Trial registration numberPROSPERO International prospective register of systematic reviews (CRD42017068915).</jats:sec

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