Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin (or ubiquitin-like protein), and remodel polyubiquitin (or ubiquitin-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five families: the UCH, USP, OTU, Josephin, and JAMM families. Four families are cysteine proteases, while the later is a family of metalloproteases. Most DUB activity is cryptic and active site rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain multiple domains with insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multi-protein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have lead to new insights into the function of yeast and human DUBs. This review will discuss ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes. Specific examples are drawn from studies of protein degradation, DNA repair, chromatin remodeling, cell cycle regulation, endocytosis, and modulation of signaling kinases.
Exposure to cocaine and amphetamine structurally reorganizes excitatory neurons in the medial and orbital prefrontal cortices (mPFC and oPFC), inducing dendritic spine proliferation in the mPFC and eliminating spines in the oPFC. Modifications may be causally associated with addiction etiology. Certain cytoskeletal regulatory proteins expressed in the oPFC and implicated in postnatal neural development also regulate behavioral sensitivity to cocaine, potentially opening a window of opportunity for the identification of novel pharmacotherapeutic targets in the treatment of drug abuse disorders. Addiction is characterized by maladaptive decision-making, a loss of control over drug consumption and habit-like drug seeking despite adverse consequences. These cognitive changes may reflect the effects of drugs of abuse on prefrontal cortical neurobiology. Here, we review evidence that amphetamine and cocaine fundamentally remodel the structure of excitatory neurons in the prefrontal cortex. We summarize evidence in particular that these psychostimulants have opposing effects in the medial and orbital prefrontal cortices ('mPFC' and 'oPFC', respectively). For example, amphetamine and cocaine increase dendrite length and spine density in the mPFC, while dendrites are impoverished and dendritic spines are eliminated in the oPFC. We will discuss evidence that certain cytoskeletal regulatory proteins expressed in the oPFC and implicated in postnatal (adolescent) neural development also regulate behavioral sensitivity to cocaine. These findings potentially open a window of opportunity for the identification of novel pharmacotherapeutic targets in the treatment of drug abuse disorders in adults, as well as in drug-vulnerable adolescent populations. Finally, we will discuss the behavioral implications of drug-related dendritic spine elimination in the oPFC, with regard to reversal learning tasks and tasks that assess the development of reward-seeking habits, both used to model aspects of addiction in rodents.
Psychosocial stress has been shown to prime peripheral innate immune cells, which take on hyper-inflammatory phenotypes and are implicated in depressive-like behavior in mouse models. However, the impact of stress on cellular metabolic states that are thought to fuel inflammatory phenotypes in immune cells are unknown. Using single cell RNA-sequencing, we investigated mRNA enrichment of immunometabolic pathways in innate immune cells of the spleen in mice subjected to repeated social defeat stress (RSDS) or no stress (NS). RSDS mice displayed a significant increase in the number of splenic macrophages and granulocytes (p < 0.05) compared to NS littermates. RSDS-upregulated genes in macrophages, monocytes, and granulocytes significantly enriched immunometabolic pathways thought to play a role in myeloid-driven inflammation (glycolysis, HIF-1 signaling, MTORC1 signaling) as well as pathways related to oxidative phosphorylation (OXPHOS) and oxidative stress (p < 0.05 and FDR<0.1). These results suggest that the metabolic enhancement reflected by upregulation of glycolytic and OXPHOS pathways may be important for cellular proliferation of splenic macrophages and granulocytes following repeated stress exposure. A better understanding of these intracellular metabolic mechanisms may ultimately help develop novel strategies to reverse the impact of stress and associated peripheral immune changes on the brain and behavior.
We recently reported that prolonged exposure to the glucocorticoid receptor (GR) ligand corticosterone impairs decision-making that is dependent on the predictive relationship between an action and its outcome (Gourley et al.; Proceedings of the National Academy of Sciences, 2012). Additionally, acute GR blockade, when paired with action-outcome conditioning, also blocks new learning. We then showed that dendritic spines in the prelimbic prefrontal cortex remodeled under both conditions. Nonetheless, the relationship between deep-layer dendritic spines and outcome-based decision-making remains opaque. We report here that a history of prolonged corticosterone exposure increases dendritic spine density in deep-layer prelimbic cortex. When spines are imaged simultaneously with corticosteroid exposure (i.e., without a washout period), dendritic spine densities are, however, reduced. Thus, the morphological response of deep-layer prelimbic cortical neurons to prolonged corticosteroid exposure may be quite dynamic, with spine elimination during a period of chronic exposure and spine proliferation during a subsequent washout period. We provide evidence, using a Rho-kinase inhibitor, that GR-mediated dendritic spine remodeling is causally related to complex decision-making. Finally, we conclude this report with evidence that a history of early-life (adolescent) GR blockade, unlike acute blockade in adulthood, enhances subsequent outcome-based decision-making. Together, our findings suggest that physiological levels of GR binding enable an organism to learn about the predictive relationship between an action and its outcome, but a history of GR blockade may, under some circumstances, also have beneficial consequences.
In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.
A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.
Fibrotic lung diseases increase with age. Previously we determined that senescence increases tissue expression of fibronectin EDA (Fn-EDA) and decreases fibroblast expression of Thy-1, and that fibrocytes contribute to fibrosis following bleomycin-induced lung injury in mice. In this study we hypothesized that fibroblasts lacking Thy-1 expression produce an extracellular matrix that promotes fibrocyte retention and myofibroblast transdifferentiation, thereby promoting fibrogenesis. Young and old mice were treated with bleomycin intratracheally; fibrocytes in the bone marrow, blood, and lungs were quantified, and lung fibroblast Thy-1 expression assessed. Bone marrow-derived fibrocytes were cultured on matrices derived from Thy-1(+) or Thy-1(−) fibroblasts ± the pro-fibrotic cytokine TGFβ1. Older mice had more fibrocytes in their bone marrows at baseline and more fibrocytes in their lungs following bleomycin treatment. In parallel, lung fibroblasts in older mice had lower expression of Thy-1 at baseline that increased transiently 7 days after bleomycin treatment but then rapidly waned such that 14 days after bleomycin treatment Thy-1 expression was again markedly lower. Fibrocytes cultured on matrices derived from Thy-1(−) fibroblasts + TGFβ1 had increased gene expression for collagen type 1, fibronectin, Fn-EDA, and α-smooth muscle actin. In parallel, whereas the matrices derived from Thy-1(−) fibroblasts stimulated phosphorylation of Akt in cultured fibrocytes, the matrices derived from Thy-1(+) fibroblasts induced apoptosis. These findings suggest that senescence increases fibrocyte recruitment to the lung following injury and that loss of Thy-1 expression by lung fibroblasts promotes fibrocyte retention and myofibroblast trans-differentiation that renders the “aging lung” susceptible to fibrosis.
Keywords: Lung Fibrosis, Thy-1, Fibrocytes, Extracellular Matrix, Fibronectin, TGF β1
Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize 'individual' poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and α-actinin, and the elongation factor, EF-1α (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a 'microdomain' where mRNA is attached and functionally expressed.
by
Xi Feng;
Frank Szulzewsky;
Alexan Yerevanian;
Zhihong Chen;
David Heinzmann;
Rikke Darling Rasmussen;
Virginia Alvarez-Garcia;
Yeonghwan Kim;
Bingcheng Wang;
Ilaria Tamagno;
Hao Zhou;
Xiaoxia Li;
Helmut Kettenmann;
Richard M. Ransohoff;
Dolores Hambardzumyan
The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/ macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G-F4/80-/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1- deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.