Showing papers in "Molecular and Cellular Biology in 2018"

Signaling Pathways Involved in the Regulation of mRNA Translation.

Abstract: Translation is a key step in the regulation of gene expression and one of the most energy-consuming processes in the cell. In response to various stimuli, multiple signaling pathways converge on the translational machinery to regulate its function. To date, the roles of phosphoinositide 3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK) pathways in the regulation of translation are among the best understood. Both pathways engage the mechanistic target of rapamycin (mTOR) to regulate a variety of components of the translational machinery. While these pathways regulate protein synthesis in homeostasis, their dysregulation results in aberrant translation leading to human diseases, including diabetes, neurological disorders, and cancer. Here we review the roles of the PI3K/AKT and MAPK pathways in the regulation of mRNA translation. We also highlight additional signaling mechanisms that have recently emerged as regulators of the translational apparatus.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis.

Abstract: Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

So Many Roads: the Multifaceted Regulation of Autophagy Induction.

Abstract: Autophagy is an evolutionary conserved, degradative process from single-cell eukaryotes, such as Saccharomyces cerevisiae, to higher mammals, such as humans. The regulation of autophagy has been elucidated through the combined study of yeast, Caenorhabditis elegans, mice, Drosophila melanogaster, and humans. MTOR, the major negative regulator of autophagy, and activating nutrient kinases, such as 5'-AMP-activated protein kinase (AMPK), interact with the autophagy regulatory complex: ULK1/2, RB1CC1, ATG13, and ATG101. The ULK1/2 complex induces autophagy by phosphorylating downstream autophagy complexes, such as the BECN1 PIK3 signaling complex that leads to the creation of LC3+ autophagosomes. We highlight in this review various reports of autophagy induction that are independent of these regulators. We discuss reports of MTOR-independent, AMPK-independent, ULK1/2-independent, and BECN1-PIK3C3-independent autophagy. We illustrate that autophagy induction and the components required vary by the nature of the induction signal and type of cell and do not always require canonical members of the autophagy signaling pathway. We illustrate that rather than thinking of autophagy as a linear pathway, it is better to think of autophagy induction as an interconnecting web of key regulators, many of which can induce autophagy through different requirements depending on the type and length of induction signals.

Role of Circular RNA DLEU2 in Human Acute Myeloid Leukemia.

Abstract: In the current study, we were interested in exploring the molecular mechanism of circular RNA DLEU2 (circRNA-DLEU2) (hsa_circ_0000488) and microRNA 496 (miR-496), as well as PRKACB, in human acute myeloid leukemia (AML) cell activities. The RNA expression levels of circRNA-DLEU2, hsa-miR-496, and PRKACB were assessed by quantitative real-time PCR (qRT-PCR). The proliferation and apoptosis abilities of the cells were determined by CCK8 assay and flow cytometry analysis. Target relationships between circRNA-DLEU2 and miR-496, as well as PRKACB, were analyzed by luciferase reporter assay and probe assay. Immunoblotting assays were used to detect the protein expression level of PRKACB. We also did in vivo experiments to observe tumor formation after overexpression of circRNA-DLEU2. Our data showed that circRNA-DLEU2 was upregulated in AML tissues and cells, which promoted AML cell proliferation and inhibited cell apoptosis. circRNA-DLEU2 promoted AML tumor formation in vivo. miR-496 was inhibited by circRNA-DLEU2 and was downregulated in AML tissues. circRNA-DLEU2 inhibited miR-496 expression and promoted PRKACB expression. miR-496 antagonized the effects of PRKACB on MOLM-13 cell proliferation and apoptosis. Collectively, circRNA-DLEU2 accelerated human AML by suppressing miR-496 and promoting PRKACB expression.

LONP1 Is Required for Maturation of a Subset of Mitochondrial Proteins, and Its Loss Elicits an Integrated Stress Response

Abstract: LONP1, an AAA+ mitochondrial protease, is implicated in protein quality control, but its precise role in this process remains poorly understood. In this study, we have investigated the role of human LONP1 in mitochondrial proteostasis and gene expression. Depletion of LONP1 resulted in partial loss of mitochondrial DNA (mtDNA) and a complete suppression of mitochondrial translation associated with impaired ribosome biogenesis. The levels of a distinct subset of mitochondrial matrix proteins (SSBP1, MTERFD3, FASTKD2, and CLPX) increased in the presence of a catalytically dead form of LONP1, suggesting that they are bona fide LONP1 substrates. Unexpectedly, the unprocessed forms of the same proteins also accumulated in an insoluble protein fraction. This subset of unprocessed matrix proteins (but not their mature forms) accumulated following depletion of the mitochondrial processing peptidase MPP, though all other MPP substrates investigated were processed normally. Prolonged depletion of LONP1 produced massive matrix protein aggregates, robustly activated the integrated stress response (ISR) pathway, and resulted in stabilization of PINK1, a mitophagy marker. These results demonstrate that LONP1 and MPPαβ are together required for the maturation of a subset of LONP1 client proteins and that LONP1 activity is essential for the maintenance of mitochondrial proteostasis and gene expression.

AMPK Inhibits ULK1-Dependent Autophagosome Formation and Lysosomal Acidification via Distinct Mechanisms.

Abstract: Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant.

Abstract: A key antioxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, posttranslational modifications (such as ubiquitination and phosphorylation), and autophagic degradation of p62/Sqstm1 and Keap1. Here we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database searches and subsequent biochemical analyses of mice revealed the presence of an mRNA that encodes a p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), formed oligomers with itself and/or the full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it in variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.

Centromere Biology: Transcription Goes on Stage.

Abstract: Accurate chromosome segregation is a fundamental process in cell biology. During mitosis, chromosomes are segregated into daughter cells through interactions between centromeres and microtubules in the mitotic spindle. Centromere domains have evolved to nucleate formation of the kinetochore, which is essential for establishing connections between chromosomal DNA and microtubules during mitosis. Centromeres are typically formed on highly repetitive DNA that is not conserved in sequence or size among organisms and can differ substantially between individuals within the same organism. However, transcription of repetitive DNA has emerged as a highly conserved property of the centromere. Recent work has shown that both the topological effect of transcription on chromatin and the nascent noncoding RNAs contribute to multiple aspects of centromere function. In this review, we discuss the fundamental aspects of centromere transcription, i.e., its dual role in chromatin remodeling/CENP-A deposition and kinetochore assembly during mitosis, from a cell cycle perspective.

CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

Abstract: We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.

53BP1 Mediates ATR-Chk1 Signaling and Protects Replication Forks under Conditions of Replication Stress.

Abstract: Complete replication of the genome is an essential prerequisite for normal cell division, but a variety of factors can block the replisome, triggering replication stress and potentially causing mutation or cell death. The cellular response to replication stress involves recruitment of proteins to stabilize the replication fork and transmit a stress signal to pause the cell cycle and allow fork restart. We find that the ubiquitously expressed DNA damage response factor 53BP1 is required for the normal response to replication stress. Using primary, ex vivo B cells, we showed that a population of 53BP1-/- cells in early S phase is hypersensitive to short-term exposure to three different agents that induce replication stress. 53BP1 localizes to a subset of replication forks following induced replication stress, and an absence of 53BP1 leads to defective ATR-Chk1-p53 signaling and caspase 3-mediated cell death. Nascent replicated DNA additionally undergoes degradation in 53BP1-/- cells. These results show that 53BP1 plays an important role in protecting replication forks during the cellular response to replication stress, in addition to the previously characterized role of 53BP1 in DNA double-strand break repair.

RACK1 Specifically Regulates Translation through Its Binding to Ribosomes

Abstract: The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

p62-Dependent Phase Separation of Patient-Derived KEAP1 Mutations and NRF2.

Abstract: Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.

O-GlcNAcylation Signal Mediates Proteasome Inhibitor Resistance in Cancer Cells by Stabilizing NRF1.

Abstract: Cancer cells often heavily depend on the ubiquitin-proteasome system (UPS) for their growth and survival. Irrespective of their strong dependence on the proteasome activity, cancer cells, except for multiple myeloma, are mostly resistant to proteasome inhibitors. A major cause of this resistance is the proteasome bounce-back response mediated by NRF1, a transcription factor that coordinately activates proteasome subunit genes. To identify new targets for efficient suppression of UPS, we explored, using immunoprecipitation and mass spectrometry, the possible existence of nuclear proteins that cooperate with NRF1 and identified O-linked N-acetylglucosamine transferase (OGT) and host cell factor C1 (HCF-1) as two proteins capable of forming a complex with NRF1. O-GlcNAcylation catalyzed by OGT was essential for NRF1 stabilization and consequent upregulation of proteasome subunit genes. Meta-analysis of breast and colorectal cancers revealed positive correlations in the relative protein abundance of OGT and proteasome subunits. OGT inhibition was effective at sensitizing cancer cells to a proteasome inhibitor both in culture cells and a xenograft mouse model. Since active O-GlcNAcylation is a feature of cancer metabolism, our study has clarified a novel linkage between cancer metabolism and UPS function and added a new regulatory axis to the regulation of the proteasome activity.

Rac1 Nanoscale Organization on the Plasma Membrane Is Driven by Lipid Binding Specificity Encoded in the Membrane Anchor.

Abstract: Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.

Regulation of Intestinal Epithelial Barrier Function by Long Noncoding RNA uc.173 through Interaction with MicroRNA 29b.

Abstract: The mammalian intestinal epithelium establishes a selectively permeable barrier that supports nutrient absorption and prevents intrusion by noxious luminal substances and microbiota. The effectiveness and integrity of the barrier function are tightly regulated via well-controlled mechanisms. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control diverse cellular processes, but their roles in the regulation of gut permeability remain largely unknown. Here we report that the T-UCR uc.173 enhances intestinal epithelial barrier function by antagonizing microRNA 29b (miR-29b). Decreasing the levels of uc.173 by gene silencing led to dysfunction of the intestinal epithelial barrier in cultured cells and increased the vulnerability of the gut barrier to septic stress in mice. uc.173 specifically stimulated translation of the tight junction (TJ) claudin-1 (CLDN1) by associating with miR-29b rather than by binding directly to CLDN1 mRNA. uc.173 acted as a natural decoy RNA for miR-29b, which interacts with CLDN1 mRNA via the 3' untranslated region and represses its translation. Ectopically expressed uc.173 abolished the association of miR-29b with CLDN1 mRNA and restored claudin-1 expression to normal levels in cells overexpressing miR-29b, thus rescuing the barrier function. These results highlight a novel function of uc.173 in controlling gut permeability and define a mechanism by which uc.173 stimulates claudin-1 translation, by decreasing the availability of miR-29b to CLDN1 mRNA.

Myc-Associated Zinc Finger Protein Regulates the Proinflammatory Response in Colitis and Colon Cancer via STAT3 Signaling.

Abstract: Myc-associated zinc finger (MAZ) is a transcription factor highly upregulated in chronic inflammatory disease and several human cancers. In the present study, we found that MAZ protein is highly expressed in human ulcerative colitis and colon cancer. However, the precise role for MAZ in the progression of colitis and colon cancer is not well defined. To determine the function of MAZ, a novel mouse model of intestinal epithelial cell-specific MAZ overexpression was generated. Expression of MAZ in intestinal epithelial cells was sufficient to enhance inflammatory injury in two complementary models of colitis. Moreover, MAZ expression increased tumorigenesis in an in vivo model of inflammation-induced colon cancer and was important for growth of human colon cancer cell lines in vitro and in vivo Mechanistically, MAZ is critical in the regulation of oncogenic STAT3 signaling. MAZ-expressing mice have enhanced STAT3 activation in the acute response to colitis. Moreover, MAZ was essential for cytokine- and bacterium-induced STAT3 signaling in colon cancer cells. Furthermore, we show that STAT3 is essential for MAZ-induced colon tumorigenesis using a chemical inhibitor. These data indicate an important functional role for MAZ in the inflammatory progression of colon cancer through regulation of STAT3 signaling and suggest that MAZ is a potential therapeutic target to dampen STAT3 signaling in colon cancer.

Asp1 Bifunctional Activity Modulates Spindle Function via Controlling Cellular Inositol Pyrophosphate Levels in Schizosaccharomyces pombe.

Abstract: The generation of two daughter cells with the same genetic information requires error-free chromosome segregation during mitosis. Chromosome transmission fidelity is dependent on spindle structure/function, which requires Asp1 in the fission yeast Schizosaccharomyces pombe Asp1 belongs to the diphosphoinositol pentakisphosphate kinase (PPIP5K)/Vip1 family which generates high-energy inositol pyrophosphate (IPP) molecules. Here, we show that Asp1 is a bifunctional enzyme in vivo: Asp1 kinase generates specific IPPs which are the substrates of the Asp1 pyrophosphatase. Intracellular levels of these IPPs directly correlate with microtubule stability: pyrophosphatase loss-of-function mutants raised Asp1-made IPP levels 2-fold, thus increasing microtubule stability, while overexpression of the pyrophosphatase decreased microtubule stability. Absence of Asp1-generated IPPs resulted in an aberrant, increased spindle association of the S. pombe kinesin-5 family member Cut7, which led to spindle collapse. Thus, chromosome transmission is controlled via intracellular IPP levels. Intriguingly, identification of the mitochondrion-associated Met10 protein as the first pyrophosphatase inhibitor revealed that IPPs also regulate mitochondrial distribution.

An Alzheimer's Disease-Linked Loss-of-Function CLN5 Variant Impairs Cathepsin D Maturation, Consistent with a Retromer Trafficking Defect.

Abstract: In a whole-exome sequencing study of multiplex Alzheimer's disease (AD) families, we investigated three neuronal ceroid lipofuscinosis genes that have been linked to retromer, an intracellular trafficking pathway associated with AD: ceroid lipofuscinosis 3 (CLN3), ceroid lipofuscinosis 5 (CLN5), and cathepsin D (CTSD). We identified a missense variant in CLN5 c.A959G (p.Asn320Ser) that segregated with AD. We find that this variant causes glycosylation defects in the expressed protein, which causes it to be retained in the endoplasmic reticulum with reduced delivery to the endolysosomal compartment, CLN5's normal cellular location. The AD-associated CLN5 variant is shown here to reduce the normal processing of cathepsin D and to decrease levels of full-length amyloid precursor protein (APP), suggestive of a defect in retromer-dependent trafficking.

MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD

Abstract: MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD -/- C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.

Growth Differentiation Factor 15 Maturation Requires Proteolytic Cleavage by PCSK3, -5, and -6.

Abstract: Growth differentiation factor 15 (GDF15) is a secreted protein with pleotropic functions from the transforming growth factor β (TGF-β) family. GDF15 is synthesized as a precursor and undergoes proteolytic cleavage to generate mature GDF15. The strong appetite-suppressing effect of mature GDF15 makes it an attractive therapeutic agent/target for diseases such as obesity and cachexia. In addition, clinical studies indicate that circulating, mature GDF15 is an independent biomarker for heart failure. We recently found that GDF15 functions as a heart-derived hormone that inhibits liver growth hormone signaling and postnatal body growth in the pediatric period. However, little is known about the mechanism of GDF15 maturation, in particular the enzymes that mediate GDF15 precursor cleavage. We investigated which candidate proteases can cleave GDF15 precursor and generate mature GDF15 in cardiomyocytes in vitro and mouse hearts in vivo We discovered that three members of the proprotein convertase, subtilisin/kexin-type (PCSK) family, namely, PCSK3, PCSK5, and PCSK6, can efficiently cleave GDF15 precursor, therefore licensing its maturation both in vitro and in vivo Our studies suggest that PCSK3, -5, and -6 mediate a crucial step of GDF15 maturation through proteolytic cleavage of the precursor. These results also reveal new targets for therapeutic application of GDF15 in treating obesity and cachexia.

Hsp70 Interacts with Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinase 2 To Regulate p38MAPK Stability and Myoblast Differentiation during Skeletal Muscle Regeneration.

Abstract: The regenerative process of injured muscle is dependent on the fusion and differentiation of myoblasts derived from muscle stem cells. Hsp70 is important for maintaining skeletal muscle homeostasis and regeneration, but the precise cellular mechanism remains elusive. In this study, we found that Hsp70 was upregulated during myoblast differentiation. Depletion or inhibition of Hsp70/Hsc70 impaired myoblast differentiation. Importantly, overexpression of p38 mitogen-activated protein kinase α (p38MAPKα) but not AKT1 rescued the impairment of myogenic differentiation in Hsp70- or Hsc70-depleted myoblasts. Moreover, Hsp70 interacted with MK2, a substrate of p38MAPK, to regulate the stability of p38MAPK. Knockdown of Hsp70 also led to downregulation of both MK2 and p38MAPK in intact muscles and during cardiotoxin-induced muscle regeneration. Hsp70 bound MK2 to regulate MK2-p38MAPK interaction in myoblasts. We subsequently identified the essential regions required for Hsp70-MK2 interaction. Functional analyses showed that MK2 is essential for both myoblast differentiation and skeletal muscle regeneration. Taken together, our findings reveal a novel role of Hsp70 in regulating myoblast differentiation by interacting with MK2 to stabilize p38MAPK.

Peroxisome Proliferator-Activated Receptor γ and Its Role in Adipocyte Homeostasis and Thiazolidinedione-Mediated Insulin Sensitization.

Abstract: Adipose tissue is a dynamic organ that makes critical contributions to whole-body metabolic homeostasis. Although recent studies have revealed that different fat depots have distinct molecular signatures, metabolic functions and adipogenic mechanisms, peroxisome proliferator-activated receptor γ (PPARγ) is still widely viewed as the master regulator of adipogenesis and critical for maintaining mature adipocyte function. Using an inducible, adipocyte-specific knockout system, we explored the role of PPARγ in mature adipocytes in vivo Short-term PPARγ deficiency in adipocytes reduces whole-body insulin sensitivity, but adipocytes are viable both in vitro and in vivo However, after exposure to a high-fat diet, even short-term PPARγ deficiency leads to rapid adipocyte death. When mature adipocytes are depleted of both PPARγ and CCAAT-enhancer-binding protein α (C/EBPα), they are rapidly depleted of lipids and undergo adipocyte death, both in vitro and in vivo Surprisingly, although thiazolidinediones (TZDs; PPARγ agonists) are thought to act mainly on PPARγ, PPARγ in adipocytes is not required for the whole-body insulin-sensitizing effect of TZDs. This offers new mechanistic aspects of PPARγ/TZD action and its effect on whole-body metabolic homeostasis.

The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation.

Abstract: Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

Analysis and Correction of Inappropriate Image Duplication: the Molecular and Cellular Biology Experience.

Abstract: We analyzed 960 papers published in Molecular and Cellular Biology (MCB) from 2009 to 2016 and found 59 (6.1%) to contain inappropriately duplicated images. The 59 instances of inappropriate image duplication led to 41 corrections, 5 retractions, and 13 instances in which no action was taken. Our experience suggests that the majority of inappropriate image duplications result from errors during figure preparation that can be remedied by correction. Nevertheless, ∼10% of papers with inappropriate image duplications in MCB were retracted (∼0.5% of total). If this proportion is representative, then as many as 35,000 papers in the literature are candidates for retraction due to inappropriate image duplication. The resolution of inappropriate image duplication concerns after publication required an average of 6 h of journal staff time per published paper. MCB instituted a pilot program to screen images of accepted papers prior to publication that identified 12 manuscripts (14.5% out of 83) with image concerns in 2 months. The screening and correction of papers before publication required an average of 30 min of staff time per problematic paper. Image screening can identify papers with problematic images prior to publication, reduces postpublication problems, and requires less staff time than the correction of problems after publication.

The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression.

Abstract: Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.

Transient Overexpression of Vascular Endothelial Growth Factor A in Adipose Tissue Promotes Energy Expenditure via Activation of the Sympathetic Nervous System.

Abstract: Adipose-derived vascular endothelial growth factor A (VEGF-A) stimulates functional blood vessel formation in obese fat pads, which in turn facilitates healthy expansion of the adipose tissue. However, the detailed mechanism(s) governing the process remains largely unknown. Here, we investigated the role of sympathetic nervous system activation in the process. To this end, we induced overexpression of VEGF-A in an adipose tissue-specific doxycycline (Dox)-inducible transgenic mouse model for a short period of time during high-fat diet (HFD) feeding. We found that local overexpression of VEGF-A in adipose tissue stimulated lipolysis and browning rapidly after Dox induction. Immunofluorescence staining against tyrosine hydroxylase (TH) indicated higher levels of sympathetic innervation in adipose tissue of transgenic mice. In response to an increased norepinephrine (NE) level, expression of β3-adrenoceptor was significantly upregulated, and the downstream protein kinase A (PKA) pathway was activated, as indicated by enhanced phosphorylation of whole PKA substrates, in particular, the hormone-sensitive lipase (HSL) in adipocytes. As a result, the adipose tissue exhibited increased lipolysis, browning, and energy expenditure. Importantly, all of these effects were abolished upon treatment with the β3-adrenoceptor antagonist SR59230A. Collectively, these results demonstrate that transient overexpressed VEGF-A activates the sympathetic nervous system, which hence promotes lipolysis and browning in adipose tissue.

The C9ORF72 Gene, Implicated in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, Encodes a Protein That Functions in Control of Endothelin and Glutamate Signaling.

Abstract: A GGGGCC repeat expansion in the C9ORF72 (C9) gene is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several mechanisms have been proposed to account for its toxicity, including the possibility that reduced C9 protein levels contribute to disease. To investigate this possibility, we examined the effects of reduced C9 levels in several cell systems. We first showed that C9 knockdown (KD) in U87 glioblastoma cells results in striking morphological changes, including vacuolization and alterations in cell size. Unexpectedly, RNA analysis revealed changes in expression of many genes, including genes involved in endothelin (EDN) signaling and immune system pathways and multiple glutamate cycling genes (e.g., EAAT2), which were verified in several cell models, including astrocytes and brain samples from C9-positive patients. Consistent with deregulation of the glutamate cycling genes, elevated intracellular glutamate was detected in both KD cells and patient astrocytes. Importantly, levels of mRNAs encoding EDN1 and its receptors, known to be elevated in ALS, were sharply increased by C9 KD, likely resulting from an observed activation of NF-κB signaling and/or a possible role of a C9 isoform in gene control.

β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination.

Abstract: The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated β1-integrin is associated with enhanced breast cancer cell survival postirradiation, but how β1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA double-strand break (DSB) repair, and we found that nonmalignant breast epithelial (S1) cells with low β1-integrin expression have a higher frequency of S-phase-specific IR-induced chromosomal aberrations than the derivative malignant breast (T4-2) cells with high β1-integrin expression. In addition, there was an increased frequency of IR-induced homologous recombination (HR) repairosome focus formation in T4-2 cells compared with that of S1 cells. Cellular levels of Rad51 in T4-2 cells, a critical factor in HR-mediated DSB repair, were significantly higher. Blocking or depleting β1-integrin activity in T4-2 cells reduced Rad51 levels, while ectopic expression of β1-integrin in S1 cells correspondingly increased Rad51 levels, suggesting that Rad51 is regulated by β1-integrin. The low level of Rad51 protein in S1 cells was found to be due to rapid degradation by the ubiquitin proteasome pathway (UPP). Furthermore, the E3 ubiquitin ligase RING1 was highly upregulated in S1 cells compared to T4-2 cells. Ectopic β1-integrin expression in S1 cells reduced RING1 levels and increased Rad51 accumulation. In contrast, β1-integrin depletion in T4-2 cells significantly increased RING1 protein levels and potentiated Rad51 ubiquitination. These data suggest for the first time that elevated levels of the extracellular matrix receptor β1-integrin can increase tumor cell radioresistance by decreasing Rad51 degradation through a RING1-mediated proteasomal pathway.

MafB Is Critical for Glucagon Production and Secretion in Mouse Pancreatic α Cells In Vivo.

Abstract: The MafB transcription factor is expressed in pancreatic α and β cells during development but becomes exclusive to α cells in adult rodents. Mafb-null (Mafb-/- ) mice were reported to have reduced α- and β-cell numbers throughout embryonic development. To further analyze the postnatal function of MafB in the pancreas, we generated endocrine cell-specific (MafbΔEndo ) and tamoxifen-dependent (MafbΔTAM ) Mafb knockout mice. MafbΔEndo mice exhibited reduced populations of insulin-positive (insulin+) and glucagon+ cells at postnatal day 0, but the insulin+ cell population recovered by 8 weeks of age. In contrast, the Arx+ glucagon+ cell fraction and glucagon expression remained decreased even in adulthood. MafbΔTAM mice, with Mafb deleted after pancreas maturation, also demonstrated diminished glucagon+ cells and glucagon content without affecting β cells. A decreased Arx+ glucagon+ cell population in MafbΔEndo mice was compensated for by an increased Arx+ pancreatic polypeptide+ cell population. Furthermore, gene expression analyses from both MafbΔEndo and MafbΔTAM islets revealed that MafB is a key regulator of glucagon expression in α cells. Finally, both mutants failed to respond to arginine, likely due to impaired arginine transporter gene expression and glucagon production ability. Taken together, our findings reveal that MafB is critical for the functional maintenance of mouse α cells in vivo, including glucagon production and secretion, as well as in development.

Mutant Isocitrate Dehydrogenase 1 Disrupts PKM2-β-Catenin-BRG1 Transcriptional Network-Driven CD47 Expression.

Abstract: A gain-of-function mutation in isocitrate dehydrogenase 1 (IDH1) affects immune surveillance in gliomas. As elevated CD47 levels are associated with immune evasion in cancers, its status in gliomas harboring mutant IDH1 (IDH1-MT cells) was investigated. Decreased CD47 expression in IDH1-R132H-overexpressing cells was accompanied by diminished nuclear β-catenin, pyruvate kinase isoform M2 (PKM2), and TCF4 levels compared to those in cells harboring wild-type IDH1 (IDH1-WT cells). The inhibition of β-catenin in IDH1-WT cells abrogated CD47 expression, β-catenin-TCF4 interaction, and the transactivational activity of β-catenin/TCF4. The reverse effect was observed in IDH1-MT cells upon the pharmacological elevation of nuclear β-catenin levels. Genetic and pharmacological manipulation of nuclear PKM2 levels in IDH1-WT and IDH1-MT cells suggested that PKM2 is a positive regulator of the β-catenin-TCF4 interaction. The Cancer Genome Atlas (TCGA) data sets indicated diminished CD47, PKM2, and β-catenin levels in IDH1-MT gliomas compared to IDH1-WT gliomas. Also, elevated BRG1 levels with mutations in the ATP-dependent chromatin-remodeling site were observed in IDH1-MT glioma. The ectopic expression of ATPase-deficient BRG1 diminished CD47 expression as well as TCF4 occupancy on its promoter. Sequential chromatin immunoprecipitation (ChIP-re-ChIP) revealed the recruitment of the PKM2-β-catenin-BRG1-TCF4 complex to the TCF4 site on the CD47 promoter. This occupancy translated into CD47 transcription, as a diminished recruitment of this complex was observed in glioma cells bearing IDH1-R132H. In addition to its involvement in CD47 transcriptional regulation, PKM2-β-catenin-BRG1 cross talk affected the phagocytosis of IDH1-MT cells by microglia.