PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes, including cell migration and chemotaxis. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium and mouse neutrophils. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor, Spt4, selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci as well as DPR proteins in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.

Lengthy trinucleotide repeats encoding polyglutamine (polyQ) stretches characterize the variant proteins of Huntington's disease and certain other inherited neurological disorders. Using a phenotypic screen to identify events that restore functionality to polyQ proteins in S. cerevisiae, we discovered that transcription elongation factor Spt4 is required to transcribe long trinucleotide repeats located either in ORFs or nonprotein-coding regions of DNA templates. Mutation of SPT4 selectively decreased synthesis of and restored enzymatic activity to expanded polyQ protein without affecting protein lacking long-polyQ stretches. RNA-seq analysis revealed limited effects of Spt4 on overall gene expression. Inhibition of Supt4h, the mammalian ortholog of Spt4, reduced mutant huntingtin protein in neuronal cells and decreased its aggregation and toxicity while not altering overall cellular mRNA synthesis. Our findings identify a cellular mechanism for transcription through repeated trinucleotides and a potential target for countermeasures against neurological disorders attributable to expanded trinucleotide regions.

Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington’s disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2’-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

von Willebrand factor (vWF) is a multimeric glycoprotein essential for hemostasis after vascular injury, which modulates platelet-surface and platelet–platelet interactions by linking platelet receptors to the extracellular matrix and to each other. The crucial role of vWF in platelet function is particularly apparent when hemodynamic conditions create blood flow with high shear stress. Through multiple functional domains, vWF mediates the attachment of platelets to exposed tissues, where immobilized vWF is able to support a homotypic and/or heterotypic self-association. The self-association of vWF is also supported by a rapidly expanding reservoir of novel evidences that the thiol/disulfide exchange regulates vWF multimer size in the blood circulation. Moreover, in addition to proteolysis and reduction of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), the regulation of vWF multimer size and self-association may depend on a disulfide bond reductase activity ascribed to thrombospondin-1 (TSP-1). Along with the classical signaling pathways in activated platelets, evidence is emerging that lipid rafts also play important roles in various phases of hemostasis and thrombosis and facilitate the interaction between the key signaling molecules. Developments in these areas will refine our understanding of the role played by vWF self-association in physiological hemostasis and pathological thrombosis.

Considerable attention has been paid to marine derived endophytic fungi, owing to their capacity to produce novel secondary metabolites with potent bioactivities. In this study, two new compounds with a prenylated diphenyl ether structure—diorcinol L (1) and (R)-diorcinol B (2)—were isolated from the marine algal-derived endophytic fungus Aspergillus tennesseensis, along with seven known compounds: (S)-diorcinol B (3), 9-acetyldiorcinol B (4), diorcinol C (5), diorcinol D (6), diorcinol E (7), diorcinol J (8), and a dihydrobenzofuran derivative 9. Their structures were elucidated by extensive NMR spectroscopy studies. Compound 2 represents the first example of an R-configuration in the prenylated moiety. All these isolated compounds were examined for antimicrobial and cytotoxic activities. Compounds 1–9 exhibited antimicrobial activities against some human- and plant-pathogenic microbes with MIC values ranging from 2 to 64 μg/mL. Moreover, compound 9 displayed considerable inhibitory activity against the THP-1 cell line in vitro, with an IC50 value of 7.0 μg/mL.

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate many activated G protein-coupled receptors (GPCRs) and play an important role in GPCR desensitization. Our previous work has demonstrated that the C-terminal conserved region (CC) of GRK-2 participates in interaction with rhodopsin and that this interaction is necessary for GRK-2-mediated receptor phosphorylation (Gan, X. Q., Wang, J. Y., Yang, Q. H., Li, Z., Liu, F., Pei, G., and Li, L. (2000) J. Biol. Chem. 275, 8469 -8474). In this report, we further investigated whether the CC of other GRKs had the same functions and defined the specific sequences in CC that are required for the functions. The CC regions of GRK-1, GRK-2, and GRK-5, representatives of the three subfamilies of GRKs, could bind rhodopsin in vitro and inhibit GRK-2-mediated phosphorylation of rhodopsin, but not a peptide GRK substrate. Through a series of mutagenesis analyses, a proline-rich motif in the CC was identified as the key element involved in the interaction between the CC region and rhodopsin. Point mutations of this motif not only disrupted the interaction of GRK-2 with rhodopsin but also abolished the ability of GRK-2 to phosphorylate rhodopsin. The findings that the CC region of GRKs interact only with the light-activated but not the non-activated rhodopsin and that the Nterminal domain of GRK-2 interacts with rhodopsin in a light-independent manner suggest that the CC region is responsible for the recognition of activated GPCRs in the canonical model.

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of ␤-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling.