Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.     

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.     

Malocclusion in an African rodent. Is it necessarily fatal?

The greater cane rat, Thryonomys swinderianus , is the second largest rodent in the southern African subregion; only the porcupine is larger. The incisors are very large and curved and their gnawing action against each other gives them chisel edges. The common belief that a broken incisor may lead to severe stress or even death because growth of the corresponding incisor, in the opposing jaw, cannot be controlled by wearing away, is not necessarily true for all rodents. At least in the greater cane rat malocclusion does not necessarily lead to the incisors entering the skull or any obvious signs of stress or suffering.     

The NF-κB RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma

High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors.     

Identification of a Sphingolipid α-Glucuronosyltransferase That Is Essential for Pollen Function in Arabidopsis

Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants.     

The Spatiotemporal Dynamics of Chromatin Protein HP1α Is Essential for Accurate Chromosome Segregation during Cell Division

Heterochromatin protein 1α (HP1α) is involved in regulation of chromatin plasticity, DNA damage repair, and centromere dynamics. HP1α detects histone dimethylation and trimethylation of Lys-9 via its chromodomain. HP1α localizes to heterochromatin in interphase cells but is liberated from chromosomal arms at the onset of mitosis. However, the structural determinants required for HP1α localization in interphase and the regulation of HP1α dynamics have remained elusive. Here we show that centromeric localization of HP1α depends on histone H3 Lys-9 trimethyltransferase SUV39H1 activity in interphase but not in mitotic cells. Surprisingly, HP1α liberates from chromosome arms in early mitosis. To test the role of this dissociation, we engineered an HP1α construct that persistently localizes to chromosome arms. Interestingly, persistent localization of HP1α to chromosome arms perturbs accurate kinetochore-microtubule attachment due to an aberrant distribution of chromosome passenger complex and Sgo1 from centromeres to chromosome arms that prevents resolution of sister chromatids. Further analyses showed that Mis14 and perhaps other PXVXL-containing proteins are involved in directing localization of HP1α to the centromere in mitosis. Taken together, our data suggest a model in which spatiotemporal dynamics of HP1α localization to centromere is governed by two distinct structural determinants. These findings reveal a previously unrecognized but essential link between HP1α-interacting molecular dynamics and chromosome plasticity in promoting accurate cell division.     

The ～16 kDa C-Terminal Sequence of Clathrin Assembly Protein AP180 Is Essential for Efficient Clathrin Binding

Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ∼16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.     

Arabidopsis OBG-Like GTPase （AtOBGL）Is Localized in Chloroplasts and Has an Essential Function in Embryo Development

OBG-like GTPases, a subfamily of P-loop GTPases, have divers and important functions in bacteria, including initiation of sporulation, DNA replication, and protein translation. Homologs of the Bacillus subtilis spo0B GTP-binding protein （OBG） can be found in plants and algae but their specific function in these organisms has not yet been elucidated. Here, it is shown that ATSG18570 encodes an Arabidopsis thaliana OBG-like protein （AtOBGL） that is localized in chlor- oplasts. In contrast to the bacterial members of this protein family, AtOBGL and other OBG-like proteins from green algae and plants possess an additional N-terminal domain, indicating functional adaptation. Disruption of the gene locus of ATOBGL by TDNA insertion resulted in an embryo-lethal phenotype and light microscopy using Normarski optics revealed that embryo maturation in the atobgl mutant is arrested at the late globular stage before development of a green embryo. Expression of 35S：：ATOBGL within the atobgl mutant background could rescue the mutant phenotype, confirming that embryo-lethality is caused by the loss of AtOBGL. Together, the data show that the bacterial-derived OBG-like GTPases have retained an essential role in chloroplasts of plants and algae. They furthermore corroborate the significance of chloroplast functions for embryo development -- an important stage within the Arabidopsis lifecycle.     

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, ²⁹⁶EEAMAIAS³⁰⁴, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.     

Strigolactone Promotes Degradation of DWARF14, an α/β Hydrolase Essential for Strigolactone Signaling in Arabidopsis

Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.     

Activation of p107 by Fibroblast Growth Factor,Which Is Essential for Chondrocyte Cell Cycle Exit,Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.