Truncated variants of MAGEL2 are involved in the etiologies of the Schaaf-Yang and Prader-Willi syndromes.

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.

A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression.

Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

Bi-allelic mutation in SEC16B alters collagen trafficking and increases ER stress.

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder characterized by bone fragility and reduced bone mass generally caused by defects in type I collagen structure or defects in proteins interacting with collagen processing. We identified a homozygous missense mutation in SEC16B in a child with vertebral fractures, leg bowing, short stature, muscular hypotonia, and bone densitometric and histomorphometric features in keeping with OI with distinct ultrastructural features. In line with the putative function of SEC16B as a regulator of trafficking between the ER and the Golgi complex, we showed that patient fibroblasts accumulated type I procollagen in the ER and exhibited a general trafficking defect at the level of the ER. Consequently, patient fibroblasts exhibited ER stress, enhanced autophagosome formation, and higher levels of apoptosis. Transfection of wild-type SEC16B into patient cells rescued the collagen trafficking. Mechanistically, we show that the defect is a consequence of reduced SEC16B expression, rather than due to alterations in protein function. These data suggest SEC16B as a recessive candidate gene for OI.

Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Hepatic inflammation elicits production of proinflammatory netrin-1 through exclusive activation of translation.

BACKGROUND AND AIMS: Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC. APPROACH AND RESULTS: A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages. CONCLUSIONS: These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation.

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties.

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington's disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide unique insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve the polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; the recruitment and accumulation of remodeled or dysfunctional membranous organelles, and the impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity.

The dead phosphatases society: a review of the emerging roles of pseudophosphatases.

Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. Research on pseudoenzymes is an emerging area of interest, with new biological functions repurposed from catalytically active relatives. Here, we provide an overview of the pseudophosphatases identified to date in all major phosphatase families. We will highlight the degeneration of the various catalytic sequence motifs and discuss the challenges associated with the experimental determination of catalytic inactivity. We will also summarize the role of pseudophosphatases in various diseases and discuss the major challenges and future directions in this field.

LTK is an ER-resident receptor tyrosine kinase that regulates secretion.

The endoplasmic reticulum (ER) is a key regulator of cellular proteostasis because it controls folding, sorting, and degradation of secretory proteins. Much has been learned about how environmentally triggered signaling pathways regulate ER function, but only little is known about local signaling at the ER. The identification of ER-resident signaling molecules will help gain a deeper understanding of the regulation of ER function and thus of proteostasis. Here, we show that leukocyte tyrosine kinase (LTK) is an ER-resident receptor tyrosine kinase. Depletion of LTK as well as its pharmacologic inhibition reduces the number of ER exit sites and slows ER-to-Golgi transport. Furthermore, we show that LTK interacts with and phosphorylates Sec12. Expression of a phosphoablating mutant of Sec12 reduces the efficiency of ER export. Thus, LTK-to-Sec12 signaling represents the first example of an ER-resident signaling module with the potential to regulate proteostasis.

STYX: a versatile pseudophosphatase.

The pseudophosphatase STYX (serine/threonine/tyrosine interacting protein) is a catalytically inactive member of the protein tyrosine phosphatase family. We perform a phylogenetic analysis of STYX and ask how far does the pseudoenzyme status of STYX reaches in evolution. Based on our previous work, we use STYX as a showcase to discuss four basic modes of action that any given pseudoenzyme may exert. Our previous work on the effect of STYX on mitogen-activated protein kinase (MAPK) signaling led us to identify two complementary modes of action. On the one hand, STYX competes with active phosphatases for binding to MAPKs. On the other hand, STYX acts as a nuclear anchor for MAPKs, affecting their nucleo-cytoplasmic shuttling. Finally, we discuss our recent work on the regulation of FBXW7 by this pseudophosphatase and how it affects the ubiquitylation and degradation of its substrates. We discuss the biological significance of this regulatory mechanism and use it as an example for the versatility of pseudoenzymes that may divert away from merely regulating their active homologs.

Incidence of secondary glaucoma after treatment of uveal melanoma with robotic radiosurgery versus brachytherapy.

PURPOSE: Different modalities of radiation therapy nowadays allow for effective treatment of uveal melanoma combined with the advantage of eye preservation. However, this advantage can secondarily be impaired by radiation-related side effects. After local recurrence, secondary glaucoma (SG) has been described as second most frequent complication leading to need of enucleation. This study compares the incidence of SG after conventional Ruthenium (Ru)-106 brachytherapy (BT) versus CyberKnife robotic radiosurgery (RRS) which has been gaining importance lately as an efficient treatment option offering improved patient comfort. METHODS: Medical records of all patients diagnosed with uveal melanoma in the Eye Clinic of the Ludwig-Maximilians-University Munich between 2007 and 2013 were reviewed. A total of 268 eyes of 268 patients treated with Ru-106 BT or CyberKnife-RRS as monotherapy were entered in this retrospective cohort study. Incidence of SG was correlated with treatment modality and baseline tumour characteristics. RESULTS: Fifty-three patients (19.8%) developed SG. At 5 years, SG was significantly more frequent after RRS (46.7%) than BT (11.1%); however, tumour thickness (maximum apical height) as a marker of tumour progress was more pronounced in the RRS group. Subgroup analysis of 178 patients for tumours amenable to both BT and RRS (thickness ≤6 mm) revealed comparable results at 3 years (RRS: 13.8 versus BT: 11.2%), but a trend towards increased incidence after RRS beyond year three. However, this difference was not significant at 5 years (28.2% versus 11.2%, p = 0.138). Tumour thickness was significantly associated with incidence of SG. CONCLUSION: In tumours ≤6 mm thickness, RRS and BT seem to offer a comparable safety profile in terms of SG. Beyond year three, SG was tendentially, but not significantly more frequent after RRS. Increasing tumour thickness is associated with risk of SG.

The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function.

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.

Regulation of Sec16 levels and dynamics links proliferation and secretion.

We currently lack a broader mechanistic understanding of the integration of the early secretory pathway with other homeostatic processes such as cell growth. Here, we explore the possibility that Sec16A, a major constituent of endoplasmic reticulum exit sites (ERES), acts as an integrator of growth factor signaling. Surprisingly, we find that Sec16A is a short-lived protein that is regulated by growth factors in a manner dependent on Egr family transcription factors. We hypothesize that Sec16A acts as a central node in a coherent feed-forward loop that detects persistent growth factor stimuli to increase ERES number. Consistent with this notion, Sec16A is also regulated by short-term growth factor treatment that leads to increased turnover of Sec16A at ERES. Finally, we demonstrate that Sec16A depletion reduces proliferation, whereas its overexpression increases proliferation. Together with our finding that growth factors regulate Sec16A levels and its dynamics on ERES, we propose that this protein acts as an integrator linking growth factor signaling and secretion. This provides a mechanistic basis for the previously proposed link between secretion and proliferation.

Day of the dead: pseudokinases and pseudophosphatases in physiology and disease.

Pseudophosphatases and pseudokinases are increasingly viewed as integral elements of signaling pathways, and there is mounting evidence that they have frequently retained the ability to interact with cellular 'substrates', and can exert important roles in different diseases. However, these pseudoenzymes have traditionally received scant attention compared to classical kinases and phosphatases. In this review we explore new findings in the emerging pseudokinase and pseudophosphatase fields, and discuss their different modes of action which include exciting new roles as scaffolds, anchors, spatial modulators, traps, and ligand-driven regulators of canonical kinases and phosphatases. Thus, it is now apparent that pseudokinases and pseudophosphatases both support and drive a panoply of signaling networks. Finally, we highlight recent evidence on their involvement in human pathologies, marking them as potential novel drug targets.

Pseudophosphatase STYX modulates cell-fate decisions and cell migration by spatiotemporal regulation of ERK1/2.

Serine/threonine/tyrosine-interacting protein (STYX) is a catalytically inactive member of the dual-specificity phosphatases (DUSPs) family. Whereas the role of DUSPs in cellular signaling is well explored, the function of STYX is still unknown. Here, we identify STYX as a spatial regulator of ERK signaling. We used predictive-model simulation to test several hypotheses for possible modes of STYX action. We show that STYX localizes to the nucleus, competes with nuclear DUSP4 for binding to ERK, and acts as a nuclear anchor that regulates ERK nuclear export. Depletion of STYX increases ERK activity in both cytosol and nucleus. Importantly, depletion of STYX causes an ERK-dependent fragmentation of the Golgi apparatus and inhibits Golgi polarization and directional cell migration. Finally, we show that overexpression of STYX reduces ERK1/2 activation, thereby blocking PC12 cell differentiation. Overall, our results identify STYX as an important regulator of ERK1/2 signaling critical for cell migration and PC12 cell differentiation.

Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection.

Shigella flexneri type III secreted effector OspF harbors a phosphothreonine lyase activity that irreversibly dephosphorylates MAP kinases (MAPKs) p38 and ERK in infected epithelial cells and thereby, dampens innate immunity. Whereas this activity has been well characterized, the impact of OspF on other host signaling pathways that control inflammation was unknown. Here we report that OspF potentiates the activation of the MAPK JNK and the transcription factor NF-κB during S. flexneri infection. This unexpected effect of OspF was dependent on the phosphothreonine lyase activity of OspF on p38, and resulted from the disruption of a negative feedback loop regulation between p38 and TGF-beta activated kinase 1 (TAK1), mediated via the phosphorylation of TAK1-binding protein 1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the impact of OspF on the activation of NF-κB, JNK and c-Jun, and demonstrate the existence of a negative feedback loop regulation between p38 and TAK1 during S. flexneri infection. Furthermore, this study validates the use of bacterial effectors as molecular tools to identify the crosstalks that connect important host signaling pathways induced upon bacterial infection.

Role of the lectin VIP36 in post-ER quality control of human alpha1-antitrypsin.

The leguminous-type (L-type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high-mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar-binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein alpha1-antitrypsin (alpha1-AT) as a cargo of VIP36. The VIP36/alpha1-AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high-mannose form of alpha1-AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of alpha1-AT were inactivated by mutagenesis. Silencing VIP36 accelerated alpha1-AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and alpha1-AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post-ER quality control of alpha1-AT.

Sec24- and ARFGAP1-dependent trafficking of GABA transporter-1 is a prerequisite for correct axonal targeting.

The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.

Actigraphy--a useful tool for motor activity monitoring in stroke patients.

AIM: The aim of the present study was the evaluation of actigraphy as a tool to objectify the recovery process after motor paresis due to stroke. METHODS: The motor activity of both arms of patients suffering from stroke was actigraphically recorded at four different time points during the course of rehabilitation: 24-36 h, 5-7 days, 3 months, and 6 months after stroke. RESULTS: Motor activity monitored by wrist-worn actigraphs located at the impaired side revealed an increase in activity between the first two time points and the subsequent ones. Additionally, actigraphic recordings showed lower total motor activity at the impaired side as compared to the nonimpaired side. A significant positive correlation was found between the actigraphically recorded motor activity and the results of the Scandinavian Stroke scale, the Barthel Index, the Rankin Scale Score and with the Motoricity Index during the 1st week, which corresponds to the time when neurological deficits were most pronounced. CONCLUSION: Our results suggest that actigraphy is a useful tool in the objective evaluation of motor activity after stroke. Moreover, actigraphy covers additional aspects that are not reflected by the usual stroke scales in a clinical situation.

Signal-dependent export of GABA transporter 1 from the ER-Golgi intermediate compartment is specified by a C-terminal motif.

The C-terminus of GABA transporter 1 (GAT1, SLC6A1) is required for trafficking of the protein through the secretory pathway to reach its final destination, i.e. the rim of the synaptic specialization. We identified a motif of three hydrophobic residues (569VMI571) that was required for export of GAT1 from the ER-Golgi intermediate compartment (ERGIC). This conclusion was based on the following observations: (i) GAT1-SSS, the mutant in which 569VMI571 was replaced by serine residues, was exported from the ER in a COPII-dependent manner but accumulated in punctate structures and failed to reach the Golgi; (ii) under appropriate conditions (imposing a block at 15 degrees C, disruption of COPI), these structures also contained ERGIC53; (iii) the punctae were part of a dynamic compartment, because it was accessible to a second anterograde cargo [the temperature-sensitive variant of vesicular stomatitis virus G protein (VSV-G)] and because GAT1-SSS could be retrieved from the punctate structures by addition of a KKxx-based retrieval motif, which supported retrograde transport to the ER. To the best of our knowledge, the VMI-motif of GAT1 provides the first example of a cargo-based motif that specifies export from the ERGIC.