Enzymatic reactions by five chalcone synthase homologs from hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

Chalcone synthase homologues from Humulus lupulus: some enzymatic properties and expression

The enzymatic properties of four chalcone synthase homologues CHS_H1, VPS, CHS 2 and CHS 4 from Humulus lupulus L. were investigated after heterologous expression in Escherichia coli. It was found that both VPS and CHS_H1 can utilize isovaleryl-CoA and isobutyryl-CoA as substrates producing compounds with positions in thin layer chromatography characteristic for phloroisovalerophenone and phloroisobutyrophenone. These reactions are accompanied by the formation of associated byproducts. The formation of naringenin chalcone can be catalyzed primarily by CHS_H1. Comparatively the ability of VPS to perform chalcone synthase reaction is very limited. Since only CHS_H1 has true chalcone synthase activity, this enzyme can be considered a key enzyme in prenylflavonoid biosynthesis. Both CHS 2 and CHS 4 utilize isovaleryl-CoA and isobutyryl-CoA as substrates, but the reactions were prematurely terminated. In comparison with VPS and CHS_H1, the optimum pH of CHS 2 was shifted to lower value. High expression of chalcone synthase-like genes were found in maturating hop cones of cultivars with high bitter acid content (Agnus, Magnum, Target) by Northern and Western blotting using probes specific for vps, chs_H1, chs 4 and polyspecific serum risen against recombinant protein CHS4, respectively. It was also found that these cultivars maintained expression of CHS homologues for a longer period of time during cone development in contrast to time-limited expression of CHS homologues in cultivars with low bitter acids content.     

Construction of gene expression system in hop (Humulus lupulus) lupulin gland using valerophenone synthase promoter

The promoter region of the valerophenone synthase (VPS) gene was isolated from hop (Humulus lupulus). VPS, a member of the chalcone synthase (CHS) super-family, catalyzes the biosynthesis reaction of the hop resin that significantly accumulates in the cone's secretory gland called the "lupulin gland". The typical H-box and G-box sequences, which exist in many plants' CHS promoters and act as cis-elements for tissue specificity, UV-light induction, etc., were not found in the isolated VPS promoter, although the H-box-like sequence (CCTTACC, CCTAACC) and the core sequence (ACGT) of the G-box were observed. The transformation experiment using the VPS promoter-UIDA gene fusion revealed that the promoter acts not only in the lupulin gland but also in the glands of leaf and stem. On the other hand, the VPS promoter activity was not induced by UV-irradiation.     

Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.     

The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization

Ubiquitination and deubiquitination regulate various cellular processes. We have recently shown that the deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM3 (AMSH3) is involved in vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. However, little is known about the identity of its interaction partners and deubiquitination substrates. Here, we provide evidence that AMSH3 interacts with ESCRT-III subunits VPS2.1 and VPS24.1. The interaction of ESCRT-III subunits with AMSH3 is mediated by the MIM1 domain and depends on the MIT domain of AMSH3. We further show that AMSH3, VPS2.1, and VPS24.1 localize to class E compartments when ESCRT-III disassembly is inhibited by coexpression of inactive Suppressor of K+ transport Defect 1 (SKD1), an AAA-ATPase involved in the disassembly of ESCRT-III. We also provide evidence that AMSH3 and SKD1 compete for binding to VPS2.1. Furthermore, we show that the loss of AMSH3 enzymatic activity leads to the formation of cellular compartments that contain AMSH3, VPS2.1, and VPS24.1. Taken together, our study presents evidence that AMSH3 interacts with classical core ESCRT-III components and thereby provides a molecular framework for the function of AMSH3 in plants.     

Essential ingredients for HIV-1 budding

HIV-1 engages the cellular ESCRT-III/VPS4 membrane scission machinery for its escape from host cells. Three papers now begin to demystify its mode of action by showing that HIV-1 requires only the transient recruitment of a surprisingly small subset of ESCRT-III components, whose membrane abscission function depends on VPS4 activity.     

Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones.

Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (alpha- and beta-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 microm for isovaleryl-CoA, 10 microm for isobutyryl-CoA and 33 microm for malonyl-CoA, were found. The amino-acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed.     

Echovirus 1 infection depends on biogenesis of novel multivesicular bodies

Non-enveloped picornavirus echovirus 1 (EV1) clusters its receptor α2β1 integrin and causes their internalization and accumulation in α2β1 integrin enriched multivesicular bodies (α2-MVBs). Our results here show that these α2-MVBs are distinct from acidic late endosomes/lysosomes by several criteria: (i) live intra-endosomal pH measurements show that α2-MVBs are not acidic, (ii) they are not positive for the late endosomal marker LBPA or Dil-LDL internalized to lysosomes, and (iii) simultaneous stimulation of epidermal growth factor receptor (EGFR) and α2β1 integrin clustering leads to their accumulation in separate endosomes. EGFR showed downregulation between 15 min and 2 h, whereas accumulation of α2β1 integrin/EV1 led to an increase of integrin fluorescence in cytoplasmic vesicles further suggesting that EV1 pathway is separate from the lysosomal downregulation pathway. In addition, the results demonstrate the involvement of ESCRTs in the biogenesis of α2-MVBs. Overexpression of dominant-negative form of VPS4 inhibited biogenesis of α2-MVBs and efficiently prevented EV1 infection. Furthermore, α2-MVBs were positive for some members of ESCRTs such as Hrs, VPS37A and VPS24 and the siRNA treatment of TSG101, VPS37A and VPS24 inhibited EV1 infection. Our results show that the non-enveloped EV1 depends on biogenesis of novel multivesicular structures for successful infection.     

Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Protozoan grazing is a major mortality factor faced by bacteria in the environment. Vibrio cholerae, the causative agent of the disease cholera, is a natural inhabitant of aquatic ecosystems, and its survival depends on its ability to respond to stresses, such as predation by heterotrophic protists. Previous results show that grazing pressure induces biofilm formation and enhances a smooth to rugose morphotypic shift, due to increased expression of Vibrio polysaccharide (VPS). In addition to negatively controlling vps genes, the global quorum sensing (QS) regulator, HapR, plays a role in grazing resistance as the ΔhapR strain is efficiently consumed while the wild type (WT) is not. Here, the relative and combined contributions of VPS and QS to grazing resistance were investigated by exposing VPS and HapR mutants and double mutants in VPS and HapR encoding genes at different phases of biofilm development to amoeboid and flagellate grazers. Data show that the WT biofilms were grazing resistant, the VPS mutants were less resistant than the WT strain, but more resistant than the QS mutant strain, and that QS contributes to grazing resistance mainly in mature biofilms. In addition, grazing effects on biofilms of mixed WT and QS mutant strains were investigated. The competitive fitness of each strain in mixed biofilms was determined by CFU and microscopy. Data show that protozoa selectively grazed the QS mutant in mixed biofilms, resulting in changes in the composition of the mixed community. A small proportion of QS mutant cells which comprised 4% of the mixed biofilm biovolume were embedded in grazing resistant WT microcolonies and shielded from predation, indicating the existence of associational protection in mixed biofilms.     

The Expression Changes of Vacuolar Protein Sorting 4B (VPS4B) Following Middle Cerebral Artery Occlusion (MCAO) in Adult Rats Brain Hippocampus

Vacuolar protein sorting 4 (VPS4), is a member of ATPases associated with diverse cellular activities protein family. VPS4 is composed of VPS4A and VPS4B, VPS4B plays an important role in the lysosomal degradation pathway, intracellular protein trafficking, virus budding and abscission of cytokinesis. However, information regarding its distribution and possible function in the central nervous system is limited. Therefore, we performed a middle cerebral artery occlusion (MCAO) in adult rats and detected the dynamic changes of VPS4B in hippocampus CA1 subregion. We found that the VPS4B expression was increased strongly after MCAO and reached the peak after 3 days. VPS4B mainly located in the cytoplasm of neurons, but not astrocytes and microglia. Moreover, there was a concomitant up-regulation of active caspase-3. In vitro studies indicated that the up-regulation of VPS4B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knock-down of VPS4B in cultured differentiated PC12 cells by siRNA showed that VPS4B promoted the expression of active caspase-3. Collectively, all these results and MTT assay suggested that the up-regulation of VPS4B played an important role in the pathophysiology after MCAO, and further research is needed to have a good understanding of its function and mechanism.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

Identification of human VPS37C, a component of endosomal sorting complex required for transport-I important for viral budding

Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.     

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p) . Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.     

PKS activities and biosynthesis of cannabinoids and flavonoids in Cannabis sativa L. plants

Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.     

PIK3C3/VPS34 control by acetylation

PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) converts phosphatidylinositol (PtdIns) to phosphatidylinositol-3-phosphate (PtdIns3P), sustaining macroautophagy/autophagy and endosomal transport. So far, facilitating the assembly of the PIK3C3/VPS34-BECN1-PIK3R4/VPS15/p150 core complex at distinct membranes is the only known way to activate PIK3C3/VPS34 in cells. We have recently revealed a novel mechanism that regulates PIK3C3/VPS34 activation; cellular PIK3C3/VPS34 is repressed under nutrient-rich conditions by EP300/p300-mediated acetylation. Following nutrient-deprivation that drops EP300 activity, PIK3C3/VPS34 is liberated by deacetylation. Intriguingly, while deacetylation of the N-terminal K29 residue accounts for core complex formation, deacetylation at the C-terminal K771 site determines the binding of PIK3C3/VPS34 to its substrate PtdIns. In vitro and in cell evidence shows that EP300-dependent acetylation and deacetylation is a switch for turning off/on PIK3C3/VPS34 in which deacetylation of K771 is required for its full activation. This PIK3C3/VPS34 activation mechanism is utilized not only by starvation-induced autophagy but also by autophagy without the involvement of AMPK, MTORC1 or ULK1. These findings suggest an alternative circuit in cells for PIK3C3/VPS34 activation, which is involved in membrane transformations in response to metabolic and nonmetabolic cues.     

Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.