Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in C. elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signaling effector that controls diverse cellular functions.

C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons.

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

Cytosolic calcium regulates cytoplasmic accumulation of TDP-43 through Calpain-A and Importin α3.

Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. Here, we identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.

Dysregulated Plasma Membrane Turnover Underlying Dendritic Pathology in Neurodegenerative Diseases.

Due to their enormous surface area compared to other cell types, neurons face unique challenges in properly handling supply and retrieval of the plasma membrane (PM)-a process termed PM turnover-in their distal areas. Because of the length and extensiveness of dendritic branches in neurons, the transport of materials needed for PM turnover from soma to distal dendrites will be inefficient and quite burdensome for somatic organelles. To meet local demands, PM turnover in dendrites most likely requires local cellular machinery, such as dendritic endocytic and secretory systems, dysregulation of which may result in dendritic pathology observed in various neurodegenerative diseases (NDs). Supporting this notion, a growing body of literature provides evidence to suggest the pathogenic contribution of dysregulated PM turnover to dendritic pathology in certain NDs. In this article, we present our perspective view that impaired dendritic endocytic and secretory systems may contribute to dendritic pathology by encumbering PM turnover in NDs.

Ataxin-2 Dysregulation Triggers a Compensatory Fragile X Mental Retardation Protein Decrease in Drosophila C4da Neurons.

Dendrites require precise and timely delivery of protein substrates to distal areas to ensure the correct morphology and function of neurons. Many of these protein substrates are supplied in the form of ribonucleoprotein (RNP) complex consisting of RNA-binding proteins (RBPs) and mRNAs, which are subsequently translated in distal dendritic areas. It remains elusive, however, whether key RBPs supply mRNA according to local demands individually or in a coordinated manner. In this study, we investigated how Drosophila sensory neurons respond to the dysregulation of a disease-associated RBP, Ataxin-2 (ATX2), which leads to dendritic defects. We found that ATX2 plays a crucial role in spacing dendritic branches for the optimal dendritic receptive fields in Drosophila class IV dendritic arborization (C4da) neurons, where both expression level and subcellular location of ATX2 contribute significantly to this effect. We showed that translational upregulation through the expression of eukaryotic translation initiation factor 4E (eIF4E) further enhanced the ATX2-induced dendritic phenotypes. Additionally, we found that the expression level of another disease-associated RBP, fragile X mental retardation protein (FMRP), decreased in both cell bodies and dendrites when neurons were faced with aberrant upregulation of ATX2. Finally, we revealed that the PAM2 motif of ATX2, which mediates its interaction with poly(A)-binding protein (PABP), is potentially necessary for the decrease of FMRP in certain neuronal stress conditions. Collectively, our data suggest that dysregulation of RBPs triggers a compensatory regulation of other functionally-overlapping RBPs to minimize RBP dysregulation-associated aberrations that hinder neuronal homeostasis in dendrites.

Doxorubicin inhibits PD-L1 expression by enhancing TTP-mediated decay of PD-L1 mRNA in cancer cells.

Recent research revealed that doxorubicin (DOX) decreased expression of programmed death-ligand 1 (PD-L1) in cancer cells. However, the detailed mechanisms underlying this effect are not well established. Here, we demonstrate that doxorubicin down-regulates PD-L1 expression through induction of AU-rich element (ARE) binding protein tristetraprolin (TTP) in cancer cells. PD-L1 mRNA contain three AREs within its 3'UTR. Doxorubicin induced expression of TTP, increased TTP binding to the 3rd ARE of the PD-L1 3'UTR, and increased decay of PD-L1 mRNA. Inhibition of TTP abrogates the inhibitory effect of doxorubicin on PD-L1 expression. Our data suggest that TTP plays a key role in doxorubicin-mediated down-regulation of PD-L1 by enhancing degradation of PD-L1 mRNA in cancer cells.

ReHypar: a recursive hybrid chunk partitioning method using NAND-flash memory SSD.

Due to the rapid development of flash memory, SSD is considered to be the replacement of HDD in the storage market. Although SSD retains several promising characteristics, such as high random I/O performance and nonvolatility, its high expense per capacity is the main obstacle in replacing HDD in all storage solutions. An alternative is to provide a hybrid structure where a small portion of SSD address space is combined with the much larger HDD address space. In such a structure, maximizing the space utilization of SSD in a cost-effective way is extremely important to generate high I/O performance. We developed ReHypar (recursive hybrid chunk partitioning) that enables improving the space utilization of SSD in the hybrid structure. The first objective of ReHypar is to mitigate the fragmentation overhead of SSD address space, by reusing the remaining free space of I/O units as much as possible. Furthermore, ReHypar allows defining several, logical data sections in SSD address space, with each of those sections being configured with the different I/O unit. We integrated ReHypar with ext2 and ext4 and evaluated it using two public benchmarks including IOzone and Postmark.

SUPPRESSOR OF VARIEGATION4, a new var2 suppressor locus, encodes a pioneer protein that is required for chloroplast biogenesis.

VAR2 is an integral thylakoid membrane protein and a member of the versatile FtsH class of metalloproteases in prokaryotes and eukaryotes. Recessive mutations in the VAR2 locus give rise to variegated plants (var2) that contain white sectors with abnormal plastids and green sectors with normal-appearing chloroplasts. In a continuing effort to isolate second-site suppressors of var2 variegation, we characterize in this report ems2505, a suppressor strain that has a virescent phenotype due to a missense mutation in At4g28590, the gene for a pioneer protein. We designated this gene SVR4 (for SUPPRESSOR OF VARIEGATION4) and the mutant allele in ems2505 as svr4-1. We demonstrate that SVR4 is located in chloroplasts and that svr4-1 single mutants are normal with respect to chloroplast anatomy and thylakoid membrane protein accumulation. However, they are modestly impaired in several aspects of photochemistry and have enhanced non-photochemical quenching (NPQ) capacity. A T-DNA insertion allele of SVR4, svr4-2, is seedling-lethal due to an early blockage of chloroplast development. We conclude that SVR4 is essential for chloroplast biogenesis, and hypothesize that SVR4 mediates some aspect of thylakoid structure or function that controls NPQ. We propose that in the suppressor strain, photoinhibitory pressure caused by a lack of VAR2 is ameliorated early in chloroplast development by enhanced NPQ capacity caused by reduced SVR4 activity. This would result in an increase in the number of chloroplasts that are able to surmount a threshold necessary to avoid photo-damage and thereby develop into functional chloroplasts.

Hemobilia from ruptured hepatic artery aneurysm in polyarteritis nodosa.

Hemobilia, in patients with the diagnosis of polyarteritis nodosa, is rare at clinical presentation and has a grave prognosis. We describe a case of massive hemobilia, due to aneurysmal rupture, in a patient with polyarteritis nodosa. A 39-year-old man was admitted to the hospital with upper abdominal pain. The patient had a history of partial small bowel resection, for intestinal infarction, about 5 years prior to this presentation. Abdominal computed tomography demonstrated multiple high attenuation areas in the bile duct and gallbladder. Hemobilia with blood seepage was visualized on endoscopic retrograde cholangiopancreatography; this bleeding stopped spontaneously. The following day, the patient developed a massive gastrointestinal bleed with resultant hypovolemic shock. Emergent hepatic angiogram revealed multiple microaneurysms; a communication was identified between a branch of the left hepatic artery and the bile duct. Hepatic arterial embolization was successfully performed. The underlying disease, polyarteritis nodosa, was managed with prednisolone and cyclophosphamide.

[Clinical experience of 48 acute toxic hepatitis patients].

BACKGROUND/AIMS: Although many individual cases of toxic hepatitis have been reported in Korea, there are few comprehensive systematic studies on acute toxic hepatitis. The first aim of this study is to investigate the frequency and clinical characteristics of acute toxic hepatitis patients. The second aim of this study is to investigate the efficacy of steroid therapy for immunoallergic idiosyncrasy. METHODS: Between March 1998 and March 2004 forty eight patients were included in this study. The medical records were reviewed retrospectively. Acute toxic hepatitis was diagnosed by score of more than 3 in RUCAM criteria. All the patients were tested for hepatitis A, B and C. Other tests included antibodies to CMV and EBV, ANA, AMA and SMA. RESULTS: Seventy-three percent of the patients were female and the mean age of the patients was 47. Twenty cases of acute toxic hepatitis (42%) were related to prescribed medications. The other causes were herbs (35%) and traditional therapeutic preparations (23%). Common symptoms were jaundice (35%), fatigue (10%), fever (9%) and abdominal pain (9%). The biochemical pattern of hepatotoxicity was divided into three groups: hepatocellular (81%), mixed (13%), and cholestatic types (6%). Three patients who have prolonged and severe jaundice were classified into immunoallergic idiosyncrasy based upon clinical and histologic findings. Prednisolone was prescribed in all three cases whose bilirubin levels had been higher than 15 mg/dL for at least 7 days. Jaundice and the laboratory findings rapidly improved within 8 days since the treatment began. CONCLUSIONS: In a demographic point of view, most patients of acute toxic hepatitis were middle aged women. Jaundice was the most commonly observed symptom. Prescribed drugs were the most common cause of acute toxic hepatitis. Although most cases of toxic hepatitis will recover with supportive care after cessation of the causative agent, steroid treatment may be helpful for the patients with severe jaundice patients who have immunoallergic idiosyncrasy.