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Monoclonal Anti-alpha-Tubulin antibody produced in mouse

RRID:AB_477582

PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading.

  • Rona G
  • Elife
  • 2018 Jul 9

Literature context:


Abstract:

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

Funding information:
  • American Cancer Society - ACS 130304-RSG-16-241-01-DMC()
  • National Institutes of Health - R01- GM057691()
  • National Institutes of Health - R01-CA076584()
  • National Institutes of Health - R01-GM057587()
  • National Institutes of Health - R21-CA187612()
  • NIEHS NIH HHS - ES07784(United States)
  • NIGMS NIH HHS - R01 GM057587()
  • The Rosztoczy Foundation - Fellowship()

Brain Somatic Mutations in MTOR Disrupt Neuronal Ciliogenesis, Leading to Focal Cortical Dyslamination.

  • Park SM
  • Neuron
  • 2018 Jun 9

Literature context:


Abstract:

Focal malformations of cortical development (FMCDs), including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are major etiologies of pediatric intractable epilepsies exhibiting cortical dyslamination. Brain somatic mutations in MTOR have recently been identified as a major genetic cause of FMCDs. However, the molecular mechanism by which these mutations lead to cortical dyslamination remains poorly understood. Here, using patient tissue, genome-edited cells, and mouse models with brain somatic mutations in MTOR, we discovered that disruption of neuronal ciliogenesis by the mutations underlies cortical dyslamination in FMCDs. We found that abnormal accumulation of OFD1 at centriolar satellites due to perturbed autophagy was responsible for the defective neuronal ciliogenesis. Additionally, we found that disrupted neuronal ciliogenesis accounted for cortical dyslamination in FMCDs by compromising Wnt signals essential for neuronal polarization. Altogether, this study describes a molecular mechanism by which brain somatic mutations in MTOR contribute to the pathogenesis of cortical dyslamination in FMCDs.

Funding information:
  • NHLBI NIH HHS - HL073284(United States)

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

  • Bellelli R
  • Mol. Cell
  • 2018 May 17

Literature context:


Abstract:

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

Funding information:
  • NIAID NIH HHS - U01 AI070499(United States)

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation.

  • Lee JH
  • Mol. Cell
  • 2018 Apr 19

Literature context:


Abstract:

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which in turn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

Funding information:
  • NCI NIH HHS - T32 CA121938(United States)

Single HIV-1 Imaging Reveals Progression of Infection through CA-Dependent Steps of Docking at the Nuclear Pore, Uncoating, and Nuclear Transport.

  • Francis AC
  • Cell Host Microbe
  • 2018 Apr 11

Literature context:


Abstract:

The HIV-1 core consists of capsid proteins (CA) surrounding viral genomic RNA. After virus-cell fusion, the core enters the cytoplasm and the capsid shell is lost through uncoating. CA loss precedes nuclear import and HIV integration into the host genome, but the timing and location of uncoating remain unclear. By visualizing single HIV-1 infection, we find that CA is required for core docking at the nuclear envelope (NE), whereas early uncoating in the cytoplasm promotes proteasomal degradation of viral complexes. Only docked cores exhibiting accelerated loss of CA at the NE enter the nucleus. Interestingly, a CA mutation (N74D) altering virus engagement of host factors involved in nuclear transport does not alter the uncoating site at the NE but reduces the nuclear penetration depth. Thus, CA protects HIV-1 complexes from degradation, mediates docking at the nuclear pore before uncoating, and determines the depth of nuclear penetration en route to integration.

Funding information:
  • NCI NIH HHS - P30 CA008748(United States)
  • NIAID NIH HHS - R01 AI129862()
  • NIGMS NIH HHS - R01 GM054787()

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

  • Cioni JM
  • Neuron
  • 2018 Mar 7

Literature context:


Abstract:

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting.

Funding information:
  • NICHD NIH HHS - R01 HD032067(United States)

Inhibition of SREBP with fatostatin does not attenuate early diabetic nephropathy in male mice.

  • Van Krieken R
  • Endocrinology
  • 2018 Feb 5

Literature context:


Abstract:

Sterol regulatory element binding protein (SREBP) is an important potential mediator of kidney fibrosis, and is known to be upregulated in diabetic nephropathy. Here we evaluate the effectiveness of SREBP inhibition as treatment for diabetic nephropathy.Type 1 diabetes was induced in uninephrectomized male CD1 mice with streptozotocin. Mice were treated with the SREBP inhibitor fatostatin for 12 weeks. At endpoint, kidney function and pathology were assessed. Fatostatin inhibited the increase of both isoforms of SREBP (1 and 2) in diabetic kidneys. Treatment attenuated basement membrane thickening, but did not improve hyperfiltration, albuminuria or kidney fibrosis in diabetic mice. Surprisingly, treatment of non-diabetic mice with fatostatin led to hyperfiltration and increased glomerular volume to levels seen in diabetic mice. This was associated with increased renal inflammation and a trend to increased renal fibrosis. Both in vivo and in cultured renal proximal tubular epithelial cells, fatostatin increased expression of the pro-inflammatory cytokine monocyte chemoattracant protein-1 (MCP-1).Thus, SREBP inhibition with fatostatin is not only ineffective in preventing diabetic nephropathy, but it also leads to kidney injury in non-diabetic mice. Further research on the efficacy of other SREBP inhibitors, and the specific roles of SREBP-1 and -2, in the treatment and pathogenesis of diabetic nephropathy need to be evaluated.

Funding information:
  • NIMH NIH HHS - T32 MH020068(United States)

A Non-canonical BCOR-PRC1.1 Complex Represses Differentiation Programs in Human ESCs.

  • Wang Z
  • Cell Stem Cell
  • 2018 Feb 1

Literature context:


Abstract:

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.

Funding information:
  • NCI NIH HHS - R01 CA071540()
  • NCRR NIH HHS - S10 RR027990()
  • NICHD NIH HHS - R01 HD084459()
  • NIDCR NIH HHS - R01DE01461301(United States)
  • NIGMS NIH HHS - R01 GM105772()
  • NIGMS NIH HHS - R01 GM107092()

Juxtanodin in retinal pigment epithelial cells: Expression and biological activities in regulating cell morphology and actin cytoskeleton organization.

  • Liang F
  • J. Comp. Neurol.
  • 2018 Feb 1

Literature context:


Abstract:

Juxtanodin (JN, also known as ermin) was initially identified as an actin cytoskeleton-related oligodendroglial protein in the rat central nervous system. It was subsequently also found in the rat olfactory neuroepithelium, especially at the apical junctional belt of the sustentacular cells. We further examined JN expression and functional roles in the retina using fluorescence histochemistry, confocal microscopy, immuno-electron microscopy, molecular biology, and cell culture. Prominent JN expression was found in the photoreceptor-supporting retinal pigment epithelium (RPE), especially in a zone corresponding to the apices of RPE cells, at the roots of the RPE microvilli, and at the base of RPE cells next to the Bruch's membrane. Partial co-localization of JN immunoreactivity with F-actin (labeled with phalloidin) was observed at the apices and bases of RPE cells. No JN was detected in other cell types of the retina. In cultured human RPE cell line ARPE-19, expression of extrinsic JN up-regulated formation of actin cytoskeleton stress fibers, caused redistribution of more F-actin fibers to the cell periphery, and promoted spreading/enlargement of transfected cells. These findings suggest possible roles of JN in RPE molecular transport, phagocytosis and formation of outer blood-retinal barrier, or possible involvement of JN expression perturbations in pathogenesis of such retinal disorders as proliferative vitreoretinopathy and age-related macular degeneration.

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

  • Simões AP
  • Cell Death Dis
  • 2018 Feb 20

Literature context:


Abstract:

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Funding information:
  • NCRR NIH HHS - 5P20-RR021940(United States)

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe.

  • Margalef P
  • Cell
  • 2018 Jan 25

Literature context:


Abstract:

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1-/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.

Funding information:
  • Wellcome Trust - 100140(United Kingdom)

A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination.

  • León-Ortiz AM
  • Mol. Cell
  • 2018 Jan 18

Literature context:


Abstract:

Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress.

Funding information:
  • NINDS NIH HHS - R01 NS054221(United States)

Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome.

  • Xu M
  • eNeuro
  • 2018 Jan 20

Literature context:


Abstract:

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). Symptoms include early developmental delay, seizures, intellectual disability, nonverbal status, autistic features, postnatal microcephaly, and progressive ataxia. Neuronal development is impaired in CS, involving defects in neuronal arborization and synaptogenesis, likely underlying diminished brain growth postnatally. In addition to neurodevelopmental defects, some reports have supported neurodegenerative pathology in CS with age. The objective of this study was to determine the nature of progressive changes in the postnatal brain in Nhe6-null mice. We examined the trajectories of brain growth and atrophy in mutant mice from birth until very old age (2 yr). We report trajectories of volume changes in the mutant that likely reflect both brain undergrowth as well as tissue loss. Reductions in volume are first apparent at 2 mo, particularly in the cerebellum, which demonstrates progressive loss of Purkinje cells (PCs). We report PC loss in two distinct Nhe6-null mouse models. More widespread reductions in tissue volumes, namely, in the hippocampus, striatum, and cortex, become apparent after 2 mo, largely reflecting delays in growth with more limited tissue losses with aging. Also, we identify pronounced glial responses, particularly in major fiber tracts such as the corpus callosum, where the density of activated astrocytes and microglia are substantially increased. The prominence of the glial response in axonal tracts suggests a primary axonopathy. Importantly, therefore, our data support both neurodevelopmental and degenerative mechanisms in the pathobiology of CS.

Funding information:
  • NHLBI NIH HHS - HL67067(United States)
  • NIMH NIH HHS - R01 MH102418()
  • NIMH NIH HHS - R01 MH105442()
  • NIMH NIH HHS - R21 MH115392()
  • NIMH NIH HHS - R25 MH101076()
  • NINDS NIH HHS - F31 NS093880()

Reversible Conformational Conversion of α-Synuclein into Toxic Assemblies by Glucosylceramide.

  • Zunke F
  • Neuron
  • 2017 Dec 20

Literature context:


Abstract:

α-Synuclein (α-syn) aggregation is a key event in Parkinson's disease (PD). Mutations in glycosphingolipid (GSL)-degrading glucocerebrosidase are risk factors for PD, indicating that disrupted GSL clearance plays a key role in α-syn aggregation. However, the mechanisms of GSL-induced aggregation are not completely understood. We document the presence of physiological α-syn conformers in human midbrain dopamine neurons and tested their contribution to the aggregation process. Pathological α-syn assembly mainly occurred through the conversion of high molecular weight (HMW) physiological α-syn conformers into compact, assembly-state intermediates by glucosylceramide (GluCer), without apparent disassembly into free monomers. This process was reversible in vitro through GluCer depletion. Reducing GSLs in PD patient neurons with and without GBA1 mutations diminished pathology and restored physiological α-syn conformers that associated with synapses. Our work indicates that GSLs control the toxic conversion of physiological α-syn conformers in a reversible manner that is amenable to therapeutic intervention by GSL reducing agents.

Funding information:
  • NCI NIH HHS - CA90668(United States)
  • NCRR NIH HHS - P20 RR017677()
  • NINDS NIH HHS - P30 NS081774()
  • NINDS NIH HHS - R01 NS092823()

Lithium improves cell viability in psychosine-treated MO3.13 human oligodendrocyte cell line via autophagy activation.

  • Del Grosso A
  • J. Neurosci. Res.
  • 2017 Nov 13

Literature context:


Abstract:

Globoid cell leukodystrophy (GLD) is a rare, rapidly progressing childhood leukodystrophy triggered by deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine; PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Here we report on autophagy in the human oligodendrocyte cell line MO3.13 treated with PSY and exploitation of Li as an autophagy modulator to rescue cell viability. We demonstrate that PSY causes upregulation of the autophagic flux at the level of autophagosome and autolysosome formation and LC3-II expression. We show that pretreatment with Li, a drug clinically used to treat bipolar disorders, can further stimulate autophagy, improving cell tolerance to PSY. This Li protective effect is found not to be linked to reduction of PSY-induced oxidative stress and might not stem from a reduction of PSY accumulation. These data provide novel information on the intracellular pathways activated during PSY-induced toxicity and suggest the autophagy pathway as a promising novel therapeutic target for ameliorating the GLD phenotype. © 2016 Wiley Periodicals, Inc.

Funding information:
  • NIMH NIH HHS - R01 MH093445(United States)

Delta-Secretase Phosphorylation by SRPK2 Enhances Its Enzymatic Activity, Provoking Pathogenesis in Alzheimer's Disease.

  • Wang ZH
  • Mol. Cell
  • 2017 Sep 7

Literature context:


Abstract:

Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

  • Ferry L
  • Mol. Cell
  • 2017 Aug 17

Literature context:


Abstract:

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.

Rhamnose-Containing Cell Wall Polymers Suppress Helical Plant Growth Independently of Microtubule Orientation.

  • Saffer AM
  • Curr. Biol.
  • 2017 Aug 7

Literature context:


Abstract:

Although specific organs in some plant species exhibit helical growth patterns of fixed or variable handedness, most plant organs are not helical. Here we report that mutations in Arabidopsis RHAMNOSE BIOSYNTHESIS 1 (RHM1) cause dramatic left-handed helical growth of petal epidermal cells, leading to left-handed twisted petals. rhm1 mutant roots also display left-handed growth. Furthermore, we find that RHM1 is required to promote epidermal cell expansion. RHM1 encodes a UDP-L-rhamnose synthase, and rhm1 mutations affect synthesis of the pectic polysaccharide rhamnogalacturonan-I. Unlike other mutants that exhibit helical growth of fixed handedness, the orientation of cortical microtubule arrays is unaltered in rhm1 mutants. Our findings reveal a novel source of left-handed plant growth caused by changes in cell wall composition that is independent of microtubule orientation. We propose that an important function of rhamnose-containing cell wall polymers is to suppress helical twisting of expanding plant cells.

Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria.

  • Vukotic M
  • Mol. Cell
  • 2017 Aug 3

Literature context:


Abstract:

Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT.

  • Gu Y
  • Mol. Cell
  • 2017 Jul 6

Literature context:


Abstract:

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

Funding information:
  • NCI NIH HHS - F31 CA186668()
  • NIGMS NIH HHS - R01 GM116897()
  • NINDS NIH HHS - R01 NS073831()

PPARγ Links BMP2 and TGFβ1 Pathways in Vascular Smooth Muscle Cells, Regulating Cell Proliferation and Glucose Metabolism.

  • Calvier L
  • Cell Metab.
  • 2017 May 2

Literature context:


Abstract:

BMP2 and TGFβ1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFβ1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFβ1-Stat3-FoxO1 and TGFβ1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFβ1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFβ1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFβ1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFβ1-Stat3-FoxO1 axis in TGFβ1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFβ1 pathways in VSMCs. PPARγ activation can be beneficial in TGFβ1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome.

Funding information:
  • NIDDK NIH HHS - R55 DK061935(United States)
  • NIMH NIH HHS - R21 MH098506(United States)

RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6.

  • Durkin CH
  • Dev. Cell
  • 2017 May 8

Literature context:


Abstract:

RhoA-mediated regulation of myosin-II activity in the actin cortex controls the ability of cells to contract and bleb during a variety of cellular processes, including cell migration and division. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many different viral infections. We now demonstrate that the vaccinia virus protein F11, which localizes to the plasma membrane, is required for ROCK-mediated cell contraction from 2 hr post infection. Curiously, F11-induced cell contraction is dependent on RhoC and not RhoA signaling to ROCK. Moreover, RhoC-driven cell contraction depends on the upstream inhibition of RhoD signaling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations with vaccinia have now demonstrated that RhoD recruits Pak6 to the plasma membrane to antagonize RhoC signaling during cell contraction and blebbing.

Mutant Huntingtin Disrupts the Nuclear Pore Complex.

  • Grima JC
  • Neuron
  • 2017 Apr 5

Literature context:


Abstract:

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

Funding information:
  • NINDS NIH HHS - R01 NS082338()
  • NINDS NIH HHS - R01 NS085207()
  • NINDS NIH HHS - R01 NS090390()
  • NINDS NIH HHS - R01 NS094239()
  • NINDS NIH HHS - U54 NS091046()

Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis.

  • Sidhaye J
  • Elife
  • 2017 Apr 4

Literature context:


Abstract:

Organ formation is a multi-scale event that involves changes at the intracellular, cellular and tissue level. Organogenesis often starts with the formation of characteristically shaped organ precursors. However, the cellular mechanisms driving organ precursor formation are often not clear. Here, using zebrafish, we investigate the epithelial rearrangements responsible for the development of the hemispherical retinal neuroepithelium (RNE), a part of the optic cup. We show that in addition to basal shrinkage of RNE cells, active migration of connected epithelial cells into the RNE is a crucial player in its formation. This cellular movement is driven by progressive cell-matrix contacts and actively translocates prospective RNE cells to their correct location before they adopt neuroepithelial fate. Failure of this migration during neuroepithelium formation leads to ectopic determination of RNE cells and consequently impairs optic cup formation. Overall, this study illustrates how spatiotemporal coordination between morphogenic movements and fate determination critically influences organogenesis.

The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover.

  • Tawo R
  • Cell
  • 2017 Apr 20

Literature context:


Abstract:

Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging.

Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

  • Sin O
  • Mol. Cell
  • 2017 Mar 16

Literature context:


Abstract:

Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.

Funding information:
  • NCATS NIH HHS - UL1 TR001863()
  • NHGRI NIH HHS - U41 HG007355()
  • NIH HHS - P40 OD010440()

GRASP1 Regulates Synaptic Plasticity and Learning through Endosomal Recycling of AMPA Receptors.

  • Chiu SL
  • Neuron
  • 2017 Mar 22

Literature context:


Abstract:

Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders.

Funding information:
  • NINDS NIH HHS - R01 NS036715()
  • NINDS NIH HHS - R01 NS073854()

Hermes Regulates Axon Sorting in the Optic Tract by Post-Trancriptional Regulation of Neuropilin 1.

  • Hörnberg H
  • J. Neurosci.
  • 2016 Dec 14

Literature context:


Abstract:

The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.

Funding information:
  • European Research Council - 322817()

Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization.

  • O'Grady GL
  • Am. J. Hum. Genet.
  • 2016 Nov 3

Literature context:


Abstract:

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis.

  • Berendzen KM
  • Cell
  • 2016 Sep 8

Literature context:


Abstract:

During neurodegenerative disease, the toxic accumulation of aggregates and misfolded proteins is often accompanied with widespread changes in peripheral metabolism, even in cells in which the aggregating protein is not present. The mechanism by which the central nervous system elicits a distal reaction to proteotoxic stress remains unknown. We hypothesized that the endocrine communication of neuronal stress plays a causative role in the changes in mitochondrial homeostasis associated with proteotoxic disease states. We find that an aggregation-prone protein expressed in the neurons of C. elegans binds to mitochondria, eliciting a global induction of a mitochondrial-specific unfolded protein response (UPR(mt)), affecting whole-animal physiology. Importantly, dense core vesicle release and secretion of the neurotransmitter serotonin is required for the signal's propagation. Collectively, these data suggest the commandeering of a nutrient sensing network to allow for cell-to-cell communication between mitochondria in response to protein folding stress in the nervous system.

Soluble Conformers of Aβ and Tau Alter Selective Proteins Governing Axonal Transport.

  • Sherman MA
  • J. Neurosci.
  • 2016 Sep 14

Literature context:


Abstract:

Despite the demonstration that amyloid-β (Aβ) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aβ and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aβ trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aβ trimers in vivo, we created a novel bigenic Tg-Aβ+Tau mouse line by crossing Tg2576 (Tg-Aβ) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aβ trimers were increased by ∼2-fold in 3-month-old Tg-Aβ and Tg-Aβ+Tau mice compared with younger mice, whereas soluble monomeric Aβ levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aβ+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aβ trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aβ and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aβ oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT: The mechanistic link between amyloid-β (Aβ) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aβ species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing Aβ and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when Aβ trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of Aβ, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that Aβ trimers might cause axonal transport deficits in AD.

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

  • Waaijers S
  • BMC Biol.
  • 2016 Aug 9

Literature context:


Abstract:

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

Funding information:
  • NIH HHS - S10 OD010737(United States)

PIAS1 Regulates Mutant Huntingtin Accumulation and Huntington's Disease-Associated Phenotypes In Vivo.

  • Ochaba J
  • Neuron
  • 2016 May 4

Literature context:


Abstract:

The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation.

FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence.

  • Franciosi F
  • Endocrinology
  • 2016 Feb 2

Literature context:


Abstract:

A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality.

Funding information:
  • Wellcome Trust - 102645(United Kingdom)

Rictor Regulates Spermatogenesis by Controlling Sertoli Cell Cytoskeletal Organization and Cell Polarity in the Mouse Testis.

  • Dong H
  • Endocrinology
  • 2015 Nov 17

Literature context:


Abstract:

Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis; however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that rapamycin-insensitive component of target of rapamycin (TOR) (Rictor), a core component of mechanistic TOR complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a cadmium chloride-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, ras-related C3 botulinum toxin substrate 1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity, and cell polarity.

Funding information:
  • NINDS NIH HHS - R37 NS041590(United States)

Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

  • Pastel E
  • Endocrinology
  • 2015 May 18

Literature context:


Abstract:

Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

Funding information:
  • NIGMS NIH HHS - R01 GM080639(United States)

Inhibitors of SCF-Skp2/Cks1 E3 ligase block estrogen-induced growth stimulation and degradation of nuclear p27kip1: therapeutic potential for endometrial cancer.

  • Pavlides SC
  • Endocrinology
  • 2013 Nov 21

Literature context:


Abstract:

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

Funding information:
  • NCRR NIH HHS - 5P20RR018788(United States)