Spindly is a nucleocytosolic O-fucosyltransferase in Dictyostelium and related proteins are widespread in protists and bacteria.

O-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium-a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galß(1,4)-GlcNAcß(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galß(1,4)-GlcNAcß(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii.

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals, O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing, and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild-type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii.

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Skp1 Dimerization Conceals Its F-Box Protein Binding Site.

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a Kd of 2.5 µM as determined by sedimentation velocity studies yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of the Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ∼750 Å2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite 1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations of ≤500 µM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit.

Skp1 is a subunit of the SCF (Skp1/Cullin 1/F-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O2-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium, where it regulates SCF assembly and O2-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in Dictyostelium, generating Galα1, 3Galα1,3Fucα1,2Galß1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [3H]glucose from UDP-[3H]glucose to the trisaccharide form of Skp1 in a glt1-dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 in glt1Δ extracts, suggesting specificity for Skp1. glt1-knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium.

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O2-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily.

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining ß strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.

Nucleocytoplasmic O-glycosylation in protists.

O-Glycosylation is an increasingly recognized modification of intracellular proteins in all kingdoms of life, and its occurrence in protists has been investigated to understand its evolution and its roles in the virulence of unicellular pathogens. We focus here on two kinds of glycoregulation found in unicellular eukaryotes: one is a simple O-fucose modification of dozens if not hundreds of Ser/Thr-rich proteins, and the other a complex pentasaccharide devoted to a single protein associated with oxygen sensing and the assembly of polyubiquitin chains. These modifications are not required for life but contingently modulate biological processes in the social amoeba Dictyostelium and the human pathogen Toxoplasma gondii, and likely occur in diverse unicellular protists. O-Glycosylation that is co-localized in the cytoplasm allows for glycoregulation over the entire life of the protein, contrary to the secretory pathway where glycosylation usually occurs before its delivery to its site of function. Here, we interpret cellular roles of nucleocytoplasmic glycans in terms of current evidence for their effects on the conformation and dynamics of protist proteins, to serve as a guide for future studies to examine their broader significance.

Role of Holmium laser enucleation of the prostate to increase cancer detection rate in patients with gray-zone PSA level.

BACKGROUND: Even though the safety of the treatment for prostate cancer diagnosed by HoLEP has been reported, the diagnostic value of HoLEP for prostate cancer detection has not been confirmed. Therefore, we investigated the diagnostic potential of HoLEP for detecting prostate cancer. METHODS: Between December 2009 and October 2015, 359 patients (median age, 70.9 years; range, 66.2-74.8) were treated simultaneously with HoLEP and transrectal prostate needle biopsy (TPNB). Of these, 199 patients with a normal digital rectal examination and serum PSA concentration between 3.5 and 10.0 ng/mL were included in the study. Univariate and multivariate logistic regression analyses were performed to identify the predictive factor for prostate cancer detected by HoLEP. RESULTS: Median PSA, prostate volume and PSA density were 4.97 ng/mL (range, 4.20-6.70), 57.40 gm (range, 43.67-77.80) and 0.09 ng/mL2 (range, 0.07-0.12), respectively. Prostate cancer (Gleason score ≥6) was detected in 46 cases (23.1%). Of these, 26 (56.5%) were detected by HoLEP pathology, 11 (23.9%) by TPNB pathology, and 9 (19.6%) by both. Univariate and multivariate logistic regression analyses were performed in 179 patients, including benign prostatic hyperplasia patients (N=153, 76.9%) and patients with cancer detected by HoLEP pathology. PSA density was identified as an independent predictor of prostate cancer detected by HoLEP in gray-zone PSA. CONCLUSIONS: HoLEP is a viable modality for detecting prostate cancer in selected cases. PSA density was an independent predictor of prostate cancer detected by HoLEP in gray-zone PSA.

Experiences with acute kidney injury complicating non-fulminant hepatitis A.

AIM: To describe the clinical features and to identify factors related to development of acute kidney injury in acute hepatitis A patients. METHODS: The study and control groups consisted of 21 and 425 patients who did or did not develop acute kidney injury, respectively, after acute hepatitis A from January 1997 to May 2007. RESULTS: There were 13 men and eight women; their mean age at diagnosis was 28.8 +/- 8.2 years in the study group. Peak values for renal and liver function impairment consisted of a median serum creatinine of 4.6 mg/dL (range, 1.5-15.3 mg/dL) on day 6 (range, days 1-20) and a median total bilirubin of 10.7 mg/dL (range, 2.6-57.5 mg/dL) on day 8 (range, day 1-19). Serum creatinine concentrations returned to baseline level by a median of 16 days and total bilirubin levels returned to normal by a median of 62 days. Six of 21 (29%) patient underwent haemodialysis. Renal biopsies performed in two patients showed acute tubular necrosis and interstitial nephritis, respectively. Logistic regression analysis showed that a lower haematocrit, the presence of coagulopathy and high C-reactive protein concentration on admission, and higher peak bilirubin value during the illness were associated with development of acute kidney injury. CONCLUSION: Acute hepatitis A should be considered in the differential diagnosis of patients with acute kidney injury, even without fulminant hepatic failure. A lower haematocrit, the presence of coagulopathy and high C-reactive protein level at presentation, and higher peak bilirubin level during the illness were associated with development of acute kidney injury in acute hepatitis A patients.

Metastatic Crohn disease clinically reminiscent of erythema nodosum on the right leg.

Cutaneous manifestations are well-recognized complications of Crohn disease (CD) that can be divided into disease-specific and reactive conditions. One of the most common reactive conditions is erythema nodosum (EN), which presents as subcutaneous tender nodules most often on the legs. On the other hand, metastatic Crohn disease (MCD) is a rare cutaneous manifestation of CD defined as the presence of noncaseating granulomas that are not contiguous with the gastrointestinal (GI) tract. The clinical presentation of MCD is variable; however, lesions often are located on the legs and genital region. We report the case of a 21-year-old woman with a 6-year history of CD who presented with MCD clinically simulating EN.