A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

The N-terminal residue influences protein stability through N-degron pathways. Here, through stability profiling of the human N-terminome, we uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 participate in the quality control of N-myristoylated proteins, wherein N-terminal glycine degrons are conditionally exposed following a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.R.T.T. is a Sir Henry Wellcome Postdoctoral Fellow (201387/Z/16/Z). Z.Z. is a Croucher Foundation Honorary PhD Scholar. This work was supported by an NIH grant (AG11085) to S.J.E. and J.W.H. S.J.E. is an Investigator with the Howard Hughes Medical Institute

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a z-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development

Changes In Threonyl-Trna Synthetase Expression And Secretion In Response To Endoplasmic Reticulum Stress By Monensin In Ovarian Cancer Cells

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery

Modulation der Unfolded Protein Response (UPR) bei Überexpression der Hepatitis-B-Oberflächenantigene unter Berücksichtigung des Transkriptionsfaktors ATF6

Eine Hepatitis-B-Infektion ist verantwortlich für ca. 350 Millionen chronisch infizierte Menschen. Mehr als 686.000 Menschen sterben an den Folgen der Infektion (Leberzirrhose/ hepatozelluläres Karzinom), weshalb die Mechanismen der intrazellulären Kommunikation dieser Infektion intensiver Forschung bedürfen. Ein direkter, intrinsischer Mechanismus der Zellschädigung bei der Hepatitis-B-Infektion beruht auf der Akkumulation der HBV-Hüllproteine (HBs) im Endoplasmatischen Retikulum (ER) der Hepatozyten, was zu Unfolded Protein Response (UPR), ER-Stress und Apoptose führen kann. Das synthetische Chaperon Phenylbuttersäure (Phenylbutyric acid (PBA)) bewirkt in verschiedenen Modellen für Protein-Aggregationskrankheiten die Auflösung zellschädigender Aggregate. Activating Transcription Factor 6 (ATF6) ist ein ER-Stress regulierender Transkriptionsfaktor. Die Akkumulation von falsch gefalteten Proteinen im ER führt zur proteolytischen Spaltung von ATF6. Der cytosolische Teil von ATF6 wird in den Zellkern transportiert und wirkt als Transkriptionsfaktor zur Bildung von Chaperonen. Das Ziel dieser Studie war die Untersuchung der Wirkung von PBA auf die Aktivierung von ATF6. Die Auswirkungen der PBA-Behandlung auf die HBs-Akkumulation und ER-Stress wurden in HBs-transgenen Mäusen analysiert. Der verknüpfende molekularbiologische Mechanismus zwischen der konsekutiven Feinverteilung sowie der intrazellulären HBs-Aggregate und der Induktion der Akute-Phase-Reaktion sollte zudem aufgeklärt werden. Es konnte gezeigt werden, dass PBA die Proteinexpression natürlicher Chaperone im Maus-Lebergewebe verringerte und die Aktivierung und Kernlokalisation des Transkriptionsfaktors ATF6 induzierte. Das synthetische Chaperon aktivierte vermutlich über den ATF6-Signalweg eine hepatische Akute-Phase-Reaktion. Die Induktion der Akute-Phase-Reaktion durch Freisetzung der Oberflächenproteine aus den Aggregaten könnte die Ursache für die fehlende therapeutische Wirkung von PBA im HBs-transgenen Maus-Modell sein. Zudem beeinflusste PBA den Energiestoffwechsel, der in diesem Zusammenhang für die Proteinfaltung als oxidativer Prozess einen relevanten Faktor darstellt. PBA kann daher nicht für die Therapie der mit HBs-Proteinaggregaten assoziierten hepatozellulären Schädigung herangezogen werden.Hepatitis-B-infection is responsible for approx. 350 million chronically infected people. More than 686.000 people die suffering from effects of the infection (liver cirrhosis/ hepatocellular carcinoma). The mechanism of intracellular communication regarding the infection requires further research. The synthetically chaperone Phenylbutyric Acid (PBA) dissolves cell-damaging aggregates in different models for protein-aggregation diseases. Accumulation of HBV envelope proteins (HBs) in the endoplasmic reticulum (ER) of hepatocytes in patients suffering from chronic HBV-infection may cause a direct, intrinsic mechanism of cell damage. Unfolded Protein Response (UPR), ER-stress and apoptosis could be the outcome of intracellular HBs-accumulation. Activating Transcription Factor 6 (ATF6) is an ER-stress regulating transcription factor. Accumulation of misfolded proteins in the ER causes proteolytic cleavage of ATF6. The cleavage product acts as a transcription factor for the formation of chaperones. The effects of PBA on HBs-accumulation and ER-stress were analyzed in HBs-transgenic mice. The aim of the present study was to investigate the effect of the chemical chaperone PBA on the activation of ATF6. It was very important to set the focus on examination the interlinking molecular biological- mechanism between the distribution of the protein aggregates and the induction of acute-phase-response. The results show that PBA reduced the protein-expression of natural chaperones in liver of HBs-transgenic mice. The activation and translocation of ATF6 to the nucleus was induced. Furthermore PBA probably activates a hepatic acute-phase- response via ATF6-signaling-pathway. The induction of acute-phase-response by releasing surface proteins from aggregates could be one reason for the missing therapeutically effect of PBA in the HBs-transgenic mouse-model. In addition, PBA influenced the energy metabolism, which represents a relevant factor for protein-folding as an oxidative process in this context. Therefore, PBA cannot be used to treat hepatocellular damage associated with HBs-protein aggregates

The analysis of programmed cell death and sporulation in Myxococcus xanthus developmental program

Coordinated behavior and the differentiation of genetic identical cells into distinct cell types have been described for several prokaryotic model species among which is Myxococcus xanthus. Upon nutrient starvation, M. xanthus undergoes a complex developmental life cycle, in which vegetative cells differentiate into environmentally resistant spores. During the developmental program the cells enter one of at least three different cell fate paths, namely “programmed cell death”, sporulation and the differentiation into a persister-like state called “peripheral rods”. The first two cell fates, “cell lysis” and “sporulation”, were examined in the current study

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a ζ-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development.This work was supported by Grant CE140100008 (BJP) and DP130102593 (CIC)

RBP-J dependent and independent signalling of EBNA-2

EBNA-2 is a multifunctional viral oncogene involved in the immortalisation of B-cells by EBV. EBNA-2 regulates transcription of viral and cellular genes in the proliferative phase of the viral life cycle, which in vitro results in the outgrowth of EBV positive B-cells into lymphoblastoid cell lines (LCLs). EBNA-2 transcriptional signalling is mediated by cellular DNA-binding proteins, such as RBP-J and PU.1, since EBNA-2 does not contain its own DNA-binding domain. In order to better characterise EBNA-2 signalling we conducted a mutational analysis of the viral LMP-1 promoter that is strongly induced by EBNA-2 in the EBV-immortalised B-cells. Our mutational analysis of the LMP-1 promoter confirmed that the PU.1 binding site is important for transactivation of the LMP-1 promoter by EBNA-2, whereas RBP-J binding to the LMP-1 promoter leads to repression and EBNA-2 binding to RBP-J is not required for transactivation. These results imply that EBNA-2 transactivates the LMP-1 promoter preferentially by an RBP-J independent mechanism. We further characterised EBNA-2 signalling by dissection of promoter targeting domains in the EBNA-2 protein. Two EBNA-2 mutants, the CR4del and WW mutant, preferentially activated RBP-J dependent and independent signalling indicating that EBNA-2 uses at least two separate signalling pathways. We introduced the characterised EBNA-2 mutants into the EBV genome and produced recombinant viruses carrying specific mutations in the EBNA-2 genes. Primary B-cells were infected with increasing titres of recombinant EBVs lacking the EBNA-2 ORF or carrying the WW or CR4del mutant. Viruses lacking the EBNA-2 ORF or carrying the WW mutant were not able to immortalise primary B-cells even at high viral titres. The CR4 region of EBNA-2 strongly influenced B-cell immortalisation efficiency and growth rate of the immortalised B-cells. These results indicate that EBNA-2 and the RBP-J signalling of EBNA-2 are absolutely essential for B-cell immortalisation by EBV. In contrast, the CR4 EBNA-2 region mediating RBP-J independent signalling is critical, but not absolutely essential for the process of EBV immortalisation

Identification of host and viral factors of arthritic alphavirus pathogenesis: the role of mannose binding lectin and the viral N-linked glycans

Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus are mosquito-borne viruses that cause epidemics of debilitating myositis and polyarthritis in humans in various areas around the world. Studies conducted in a mouse model of RRV-induced disease have demonstrated a critical role for the inflammatory response in the development of disease. In particular, the host complement system contributes significantly to damage within target tissues through activation of CR3-bearing inflammatory cells. However, the precise mechanism and ligands leading to complement activation and disease following RRV infection are not known. In these studies, we have identified critical roles for the host innate immune protein mannose binding lectin (MBL) and the viral N-linked glycans in mediating complement activation and disease following RRV infection. Using mice deficient in MBL, we demonstrated that the MBL activation pathway of the host complement system is the primary pathway required for complement activation and disease following infection. MBL recognizes and binds to terminal carbohydrates, such as mannose found on glycosylated viral proteins or on infected cells. The RRV E2 envelope glycoprotein contains two N-linked glycosylation sites that are glycosylated with a combination of high mannose and complex glycans when replicating in mammalian cells. We hypothesized that MBL recognizes the E2 N-linked glycans to activate the complement system, leading to disease. Using a panel of RRV mutants lacking one or more envelope glycans, we have found that the RRV E2 N-linked glycans contribute to MBL binding to RRV infected cells and development of disease. Viruses lacking either E2 N-linked glycosylation sites cause reduced disease in mice, while a virus lacking both sites causes very mild disease. In addition, the role of the E2 glycans is independent of replication within host tissues and recruitment of inflammatory cells. Rather, the E2 glycans were required for MBL deposition and complement activation within target tissues in vivo. These results suggest that interactions between the viral N-linked glycans and the MBL pathway play a central role in development of severe alphavirus-induced arthritis and may be an effective target for therapeutic treatment in patients infected with RRV.Doctor of Philosoph

Membrane-bound Matrix Metalloproteinases Influence Reactive Synaptogenesis Following Traumatic Brain Injury

Traumatic brain injury (TBI) produces axonal damage and deafferentation, triggering injury-induced synaptogenesis, a process influenced by matrix metalloproteinases (MMP) and their substrates. Here we report results of studies examining the expression and potential role of two membrane-bound MMPs, membrane-type 5-MMP (MT5-MMP) and a disintegrin and metalloproteinase-10 (ADAM-10), along with their common synaptic substrate N-cadherin, during the period of reactive synaptogenesis. Protein and mRNA expression of MT5-MMP, ADAM-10 and N-cadherin were compared in two TBI models, one exhibiting adaptive plasticity (unilateral entorhinal cortex lesion; UEC) and the other maladaptive plasticity (fluid percussion injury + bilateral EC lesions; TBI+BEC), targeting 2, 7, and 15d postinjury intervals. In adaptive UEC plasticity, membrane-bound MMP expression was elevated during synaptic degeneration (2d) and regeneration (7d), and normalized at 15d. By contrast, N-cadherin expression was significantly decreased at 2 and 7d after UEC, but increased during 15d synaptic stabilization. In maladaptive plasticity, 2d membrane-bound MMP expression was dampened compared to UEC, with persistent ADAM-10 elevation and reduced N-cadherin protein level at 15d. These results were supported by 7d microarray and qRT-PCR analyses, which showed transcript shifts in both hippocampus and dentate molecular layer (ML) for each model. Parallel immunohistochemistry revealed significant MT5-MMP, ADAM-10 and N-cadherin localization within ML reactive astrocytes, suggesting a glial synthetic or phagocytotic role for their processing during recovery. We also investigated the effect of MMP inhibition on molecular, electrophysiological, behavioral and structural outcome at 15d following TBI+BEC. MMP inhibitor GM6001 was administered at 6 and 7d postinjury, during elevated MT5-MMP/ADAM-10 expression and synapse regeneration. MMP inhibition showed: 1) reduced ADAM-10 and elevated N-cadherin protein expression, generating profiles similar to 15d post-UEC, 2) attenuation of deficits in the initiation phase of long-term potentiation, and 3) improved hippocampal dendritic and synaptic ultrastructure. Collectively, our results provide evidence that membrane-bound MMPs and N-cadherin influence both adaptive and maladaptive plasticity in a time and injury-dependent manner. Inhibition of membrane-bound MMPs during maladaptive plasticity produces more adaptive conditions, improving synaptic efficacy and structure. Thus, targeting MMP function and expression have potential to translate maladaptive plasticity into an adaptive process, facilitating improved recovery