Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings: Distinct cytoplasmic rods (,3–10 mm in length) and rings (,2–5 mm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in.95 % of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (.95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin

A trehalose biosynthetic enzyme doubles as an osmotic stress sensor to regulate bacterial morphogenesis

The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter's enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments

Evidence for Loss of a Partial Flagellar Glycolytic Pathway during Trypanosomatid Evolution

Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed

A controlled study of supplementation with essential amino acids and α-keto acids in the conservative management of patients with chronic renal failure

Art und Zusammensetzung einer optimalen eiweißarmen Ernährung für Patienten mit Niereninsuffizienz sind weiterhin umstritten. Die orale medikamentöse Behandlung mit essentiellen Aminosäuren oder α-Ketosäuren wird häufig empfohlen. Unsere Untersuchungen vergleichen nacheinander bei 15 ambulanten Patienten mit chronischem Nierenversagen (mittlere Kreatinin-Clearance 10,8 ml/min) unter einer eiweißarmen Ernährung von 0,57 g/kg Körpergewicht (40 g/70 kg) die Wirkung einer Substitution mit essentiellen Aminosäuren, danach die Substitution mit α-Ketosäuren gegenüber Plazebo. Der nachgewiesene Proteingehalt in der Nahrung betrug 0,55 g/kg, die Energiezufuhr 27 kcal/kg Körpergewicht, wie mehrfach Ernährungsprotokolle über jeweils 7 Tage bei den Patienten zeigen ließen. Nach einer Vorperiode von 6 Wochen nur unter diätetischen Maßnahmen erhielten alle Patienten zusätzlich 0,112 g essentielle Aminosäuren/kg Körpergewicht über 6 Wochen, danach in einer Doppelblinduntersuchung 0,105 g α-Ketosäuren/kg Körpergewicht im Vergleich gegenüber Plazebo, ebenfalls jeweils über 6 Wochen. Nüchtern-Blutuntersuchungen wurden für ein Standard-Laborwertprogramm, insbesondere für 15 Proteinmangelparameter, alle 3 Wochen durchgeführt, ferner anthropometrische und klinische Kontrollen. Die Laborwerte erbrachten keine Hinweise auf einen manifesten Proteinmangel. Die Therapie mit α-Ketosäuren erniedrigte die Phosphatspiegel signifikant (p<0,05). Dagegen konnten weder unter essentiellen Aminosäuren oder α-Ketosäuren andere für den Patienten wesentliche Effekte nachgewiesen werden. Deshalb erscheint uns eine Substitution mit essentiellen Aminosäuren oder Ketosäuren überflüssig bei Patienten mit einer chronischen Niereninsuffizienz, die sich in einem stabilen Stoffwechselgleichgewicht befinden und mit einer Eiweißzufuhr von 0,55 g/kg Körpergewicht behandelt werden. Oral therapy with essential amino acids (EAA) or α-keto acids (α-KA) has been recommended in patients with renal failure, but quality and quantity of optimal protein intake are still controversial. This study compares sequentially the effect of supplementation with EAA, and with α-KA versus placebo in 15 ambulatory patients with chronic renal failure (average creatinine clearance 10.8 ml/min), maintained on a protein diet of 0.57 g/kg body weight (40 g for a 70-kg patient). The actual dietary intake averaged 0.55 g protein/kg and 27 kcal/kg according to repeated 7-day dietary recordings. After a 6-week baseline period on this diet, all patients received additionally 0.112 g EAA/kg for 6 weeks followed by a double-blind cross-over study of 0.105 g α-KA/kg versus placebo supplementation for 6 weeks each. Fasting blood samples for multiple parameters, including 15 indicators for protein deficiency, as well as anthropometric and clinical data were evaluated every 3 weeks. Laboratory data revealed no indications of protein deficiency. Therapy with α-KA diminished serum phosphate concentration (p<0.05), however no other significant beneficial effects could be demonstrated during supplementation with either EAA or α-KA. Therefore, such supplementation to a 0.55-g/kg-protein diet appears superfluous in stable ambulatory patients with renal insufficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41743/1/394_2005_Article_BF02020747.pd

Common regulatory control of CTP synthase enzyme activity and filament formation

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity