ABSTRACT
Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.
Subject(s)
Dolichols , Dolichols/metabolism , Dolichols/biosynthesis , Humans , Glycosylation , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/metabolism , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/genetics , Male , Mutation, Missense , FemaleABSTRACT
Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/Mn2+ transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn2+ sensitivity. Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn2+ sensitivity.
Subject(s)
Antiporters/physiology , Cation Transport Proteins/physiology , Golgi Apparatus/metabolism , Manganese/metabolism , Calcium/metabolism , Congenital Disorders of Glycosylation/metabolism , Glycosylation , HEK293 Cells , Humans , Ion TransportABSTRACT
The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca2+/Mn2+ homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca2+ concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn2+ confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca2+. The use of Pmr1p mutants either defective for Ca2+ or Mn2+ transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca2+ requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn2+ inside the Golgi lumen when Pmr1p exclusively transports Ca2+. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn2+ sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn2+/Ca2+transport.
Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Golgi Apparatus/metabolism , Manganese/metabolism , Mannans/metabolism , Protein Processing, Post-Translational/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Binding Sites , Calcium Channels/chemistry , Calcium Channels/genetics , Calcium-Transporting ATPases/metabolism , Conserved Sequence , Glycosylation , Ion Transport , Molecular Chaperones/metabolism , Monosaccharides/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.
Subject(s)
Golgi Apparatus/drug effects , Lysosomes/drug effects , Manganese/pharmacology , Membrane Proteins/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Antiporters , Blotting, Western , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Cation Transport Proteins , Dose-Response Relationship, Drug , Gene Knockdown Techniques , Glutamates/genetics , Glutamates/metabolism , Glycosylation/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Membrane Proteins/genetics , Microscopy, Confocal , Mutation , Proteolysis/drug effectsABSTRACT
BACKGROUND: Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca2+/H+ antiporters regulating Ca2+ and pH homeostasis and mainly localized in the Golgi apparatus. METHODS: RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. RESULTS: In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. GENERAL SIGNIFICANCE: The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations.
Subject(s)
Alternative Splicing/genetics , Congenital Disorders of Glycosylation/genetics , Genetic Variation/genetics , Membrane Proteins/genetics , Amino Acid Sequence , Antiporters , Brain/metabolism , Calcium/metabolism , Cation Transport Proteins , Cell Line, Tumor , Endoplasmic Reticulum/genetics , Glycosylation , Golgi Apparatus/genetics , HeLa Cells , Humans , Protein Isoforms/genetics , RNA, Messenger/geneticsABSTRACT
Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.
Subject(s)
Fungal Proteins/genetics , Golgi Apparatus/physiology , Manganese/pharmacology , Membrane Proteins/deficiency , Mutation , Antiporters , Cation Transport Proteins , Congenital Disorders of Glycosylation/genetics , Fungal Proteins/metabolism , Glycosylation/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Manganese/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases in vitro, and this requires the presence of an upstream -5 histidine residue. We further show that these sites are phosphorylated in vivo and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.
Subject(s)
Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line, Tumor , Humans , Phosphorylation , Protein Binding , Protein Isoforms/metabolism , Serine/metabolismABSTRACT
TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1âµ. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Point Mutation , Antiporters , Cation Transport Proteins , Cell Membrane/genetics , Cell Membrane/metabolism , Endosomes/genetics , Endosomes/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Humans , Lysosomes/genetics , Lysosomes/metabolism , Membrane Proteins/chemistry , Protein Sorting Signals , Protein TransportABSTRACT
The modification of intracellular proteins by ubiquitin (Ub) and ubiquitin-like (UbL) proteins is a central mechanism for regulating and fine-tuning all cellular processes. Indeed, these modifications are widely used to control the stability, activity and localisation of many key proteins and, therefore, they are instrumental in regulating cellular functions as diverse as protein degradation, cell signalling, vesicle trafficking and immune response. It is thus no surprise that pathogens in general, and viruses in particular, have developed multiple strategies to either counteract or exploit the complex mechanisms mediated by the Ub and UbL protein conjugation pathways. The aim of this review is to provide an overview on the intricate and conflicting relationships that intimately link HIV-1 and these sophisticated systems of post-translational modifications.
Subject(s)
HIV Infections/physiopathology , HIV-1/metabolism , Protein Processing, Post-Translational , Ubiquitin/metabolism , Ubiquitins/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cell Nucleus/virology , Cytoplasm/metabolism , Humans , Ubiquitin/genetics , Ubiquitins/geneticsABSTRACT
Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.
Subject(s)
Gene Expression Regulation/immunology , Histocompatibility Antigens Class I/biosynthesis , Leukemia, B-Cell/immunology , MAP Kinase Signaling System/immunology , Mitogen-Activated Protein Kinase 7/physiology , Oxidative Phosphorylation , Adenosine Triphosphate/biosynthesis , Animals , Cell Line, Tumor , Cell Proliferation , Cell Survival/immunology , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Down-Regulation/immunology , Glutamine/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Jurkat Cells , Leukemia L1210 , Leukemia, B-Cell/enzymology , Leukemia, B-Cell/pathology , MAP Kinase Signaling System/genetics , Mice , Mitogen-Activated Protein Kinase 7/antagonists & inhibitors , Mitogen-Activated Protein Kinase 7/genetics , Up-Regulation/immunologyABSTRACT
The discovery of the proteasome in the late 80's as the core protease of what will be then called the ubiquitin-proteasome system, rapidly followed by the development of specific inhibitors of this enzyme, opened up a new era in biology in the 90's. Indeed, the first proteasome inhibitors were instrumental for understanding that the proteasome is a key actor in most, if not all, cellular processes. The recognition of the central role of this complex in intracellular proteolysis in turn fuelled an intense quest for novel compounds with both increased selectivity towards the proteasome and better bioavailability that could be used in fundamental research or in the clinic. To date, a plethora of molecules that target the proteasome have been identified or designed. The success of the proteasome inhibitor bortezomib (Velcade(®)) as a new drug for the treatment of Multiple Myeloma, and the ongoing clinical trials to evaluate the effect of several other proteasome inhibitors in various human pathologies, illustrate the interest for human health of these compounds.
Subject(s)
Protease Inhibitors/pharmacology , Proteasome Inhibitors , Animals , Humans , Peptides/chemistry , Peptides/pharmacology , Peptidomimetics/chemistry , Peptidomimetics/pharmacology , Protease Inhibitors/chemistry , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolismABSTRACT
Lte1 is a mitotic regulator long envisaged as a guanosine nucleotide exchange factor (GEF) for Tem1, the small guanosine triphosphatase governing activity of the Saccharomyces cerevisiae mitotic exit network. We demonstrate that this model requires reevaluation. No GEF activity was detectable in vitro, and mutational analysis of Lte1's putative GEF domain indicated that Lte1 activity relies on interaction with Ras for localization at the bud cortex rather than providing nucleotide exchange. Instead, we found that Lte1 can determine the subcellular localization of Bfa1 at spindle pole bodies (SPBs). Under conditions in which Lte1 is essential, Lte1 promoted the loss of Bfa1 from the maternal SPB. Moreover, in cells with a misaligned spindle, mislocalization of Lte1 in the mother cell promoted loss of Bfa1 from one SPB and allowed bypass of the spindle position checkpoint. We observed that lte1 mutants display aberrant localization of the polarity cap, which is the organizer of the actin cytoskeleton. We propose that Lte1's role in cell polarization underlies its contribution to mitotic regulation.
Subject(s)
Cytoskeletal Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Monomeric GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Cell Cycle/genetics , Cell Polarity/genetics , Cytoskeletal Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/physiology , Intracellular Space/genetics , Intracellular Space/metabolism , Mitosis/genetics , Monomeric GTP-Binding Proteins/genetics , Point Mutation , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Nucleic Acid , Spindle Apparatus/genetics , Spindle Apparatus/metabolismABSTRACT
Tumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine.
Subject(s)
Cancer Vaccines/immunology , Gene Knockdown Techniques , Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/immunology , Leukemia L1210/prevention & control , Lymphocyte Activation/immunology , Mitogen-Activated Protein Kinase 7/antagonists & inhibitors , Mitogen-Activated Protein Kinase 7/genetics , Animals , Cancer Vaccines/administration & dosage , Cancer Vaccines/genetics , Cell Line, Tumor , Cells, Cultured , Cytotoxicity, Immunologic/genetics , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Humans , Jurkat Cells , Killer Cells, Natural/metabolism , Leukemia L1210/enzymology , Leukemia L1210/genetics , Leukemia L1210/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 7/biosynthesis , RNA, Small Interfering/physiology , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
Whereas early cytokinesis events have been relatively well studied, little is known about its final stage, abscission. The Cdc14 phosphatase is involved in the regulation of multiple cell cycle events, and in all systems studied Cdc14 misexpression leads to cytokinesis defects. In this work, we have cloned two CDC14 cDNA from Xenopus, including a previously unreported CDC14B homologue. We use Xenopus and human cell lines and demonstrate that localization of Cdc14 proteins is independent of both cell-type and species specificity. Ectopically expressed XCdc14A is centrosomal in interphase and localizes to the midbody in cytokinesis. By using XCdc14A misregulation, we confirm its control over different cell cycle events and unravel new functions during abscission. XCdc14A regulates the G1/S and G2/M transitions. We show that Cdc25 is an in vitro substrate for XCdc14A and might be its target at the G2/M transition. Upregulated wild-type or phosphatase-dead XCdc14A arrest cells in a late stage of cytokinesis, connected by thin cytoplasmic bridges. It does not interfere with central spindle formation, nor with the relocalization of passenger protein and centralspindlin complexes to the midbody. We demonstrate that XCdc14A upregulation prevents targeting of exocyst and SNARE complexes to the midbody, both essential for abscission to occur.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle , Centrioles/metabolism , Gene Expression Regulation, Enzymologic , Xenopus Proteins/metabolism , Xenopus/genetics , Actins/physiology , Animals , Cell Cycle Proteins/physiology , Cell Line , Centrioles/physiology , Cloning, Molecular , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Microtubules/physiology , SNARE Proteins/metabolism , Xenopus/physiology , Xenopus Proteins/genetics , Xenopus Proteins/physiology , cdc25 Phosphatases/metabolismABSTRACT
The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Delta mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Delta mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions.