Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

IntroductionRegional citrate anticoagulation (RCA) for continuous renal replacement therapy is widely used in intensive care units (ICUs). However, concern exists about the safety of citrate in patients with liver failure (LF). The aim of this study was to evaluate safety and efficacy of RCA in ICU patients with varying degrees of impaired liver function.MethodsIn a multicenter, prospective, observational study, 133 patients who were treated with RCA and continuous venovenous hemodialysis (RCA-CVVHD) were included. Endpoints for safety were severe acidosis or alkalosis (pH ≤7.2 or ≥7.55, respectively) and severe hypo- or hypercalcemia (ionized calcium ≤0.9 or ≥1.5 mmol/L, respectively) of any cause. The endpoint for efficacy was filter lifetime. For analysis, patients were stratified into three predefined liver function or LF groups according to their baseline serum bilirubin level (normal liver function ≤2 mg/dl, mild LF >2 to ≤7 mg/dl, severe LF >7 mg/dl).ResultsWe included 48 patients with normal liver function, 43 with mild LF, and 42 with severe LF. LF was predominantly due to ischemia (39 %) or multiple organ dysfunction syndrome (27 %). The frequency of safety endpoints in the three patient strata did not differ: severe alkalosis (normal liver function 2 %, mild LF 0 %, severe LF 5 %; p = 0.41), severe acidosis (normal liver function 13 %, mild LF 16 %, severe LF 14 %; p = 0.95), severe hypocalcemia (normal liver function 8 %, mild LF 14 %, severe LF 12 %; p = 0.70), and severe hypercalcemia (0 % in all strata). Only three patients showed signs of impaired citrate metabolism. Overall filter patency was 49 % at 72 h. After censoring for stop of the treatment due to non-clotting causes, estimated 72-h filter survival was 96 %.ConclusionsRCA-CVVHD can be safely used in patients with LF. The technique yields excellent filter patency and thus can be recommended as first-line anticoagulation for the majority of ICU patients.Trial registrationISRCTN Registry identifier: ISRCTN92716512. Date assigned: 4 December 2008.

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The human guanylate binding protein 5 (hGBP5) belongs to the family of interferon‐γ‐inducible large GTPases, which are well known for their high induction by pro‐inflammatory cytokines. The cellular role of this protein family is unclear at this point, but there are indications for antiviral and antibacterial activity of hGBP1. hGBP5 exists in three splice variants, forming two different proteins, of which the tumor‐specific one is C‐terminally truncated by 97 amino acids, and therefore lacks the CaaX motif for geranylgeranylation. Here we present biochemical data on the splice variants of hGBP5. We show that, unlike hGBP1, hGBP5a/b and hGBP5ta do not bind GMP or produce any GMP during hydrolysis despite the fact the residues involved in GMP production from hGBP1 are conserved in hGBP5. Hydrolysis of GTP is concentration‐dependent and shows weak self‐activation. Thermodynamic studies showed strongly negative entropic changes during nucleotide binding, which reflect structural ordering in the protein during nucleotide binding. These structural changes were also observed during changes in the oligomerization state. We observed only a minor influence of the C‐terminal truncation on hydrolysis, nucleotide binding and oligomerization of hGBP5. Based on these similarities we speculate that the missing C‐terminal part, which also carries the geranylgeranylation motif, is the reason for the dysregulation of hGBP5′s function in lymphoma cells. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7555035, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7555053: hGBP5 a/b (uniprotkb:http://www.ebi.uniprot.org/entry/Q96PP8) and hGBP5 a/b (uniprotkb:http://www.ebi.uniprot.org/entry/Q96PP8) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7555028, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7555044: hGBP5ta (uniprotkb:http://www.ebi.uniprot.org/entry/Q86TM5) and hGBP5ta (uniprotkb:http://www.ebi.uniprot.org/entry/Q86TM5) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)