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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Synopsis
Mapping the interactome of the enigmatic pseudophosphatase STYX reveals it as a potentially general new regulator of SCF‐type cullin–RING ubiquitin ligases, which stabilizes substrates by blocking recruitment of F‐box‐containing adaptors.
Mass spectrometry identifies several F‐box proteins as STYX interactors.
STYX competes with the SCF subunit SKP1 for binding to the F‐box domain of FBXW7.
STYX negatively regulates FBXW7 function and stabilizes FBXW7 substrates.
High STYX and low FBXW7 levels correlate with poor relapse‐free survival of breast cancer patients.
STYX‐mediated stabilization of ubiquitination targets by blocking substrate adaptor recruitment into SCF E3s may represent a general new mode of cullin–RING ligase regulation.
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.
Synopsis
The newly found mutant SEC16B causes type I collagen accumulation in the ER. This in turn leads to increased ER stress, autophagosome numbers and enhanced apoptosis. In addition, there is alteration of secreted COL1A1 in the SEC16B mutated cells.
SEC16B homozygous mutation was identified in a boy with moderate osteogenesis imperfecta.
The mutation reduces the expression of SEC16B mRNA.
SEC16B mutant cells accumulate type I procollagen in the ER due to a secretory trafficking defect.
SEC16B is a candidate gene for osteogenesis imperfecta.
This study uncovers a new role for the SEC16B protein in the ER to Golgi Apparatus trafficking.
The newly found mutant SEC16B causes type I collagen accumulation in the ER. This in turn leads to increased ER stress, autophagosome numbers and enhanced apoptosis. In addition, there is alteration of secreted COL1A1 in the SEC16B mutated cells.
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.
Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of ...identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.
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 α1-antitrypsin (α1-AT) as a cargo of VIP36. The VIP36/α1-AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high-mannose form of α1-AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1-AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1-AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1-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 α1-AT.
Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic
reticulum (ER), because quality control is stringent. This is also true ...for G protein-coupled receptors. Here, we identify
the deubiquitinating enzyme Usp4 as an interaction partner of the A 2a adenosine receptor, a G s -coupled receptor. Usp4 binds to the carboxyl terminus of the A 2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface
expression of functionally active receptor. The effect of Usp4 on the A 2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked
by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate
receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show
that deubiquinating enzymes can regulate quality control in the ER.
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.
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.
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.
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.
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