Structural mutation analysis of PTEN and its genotype‐phenotype correlations in endometriosis and cancer

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene encodes a tumor suppressor phosphatase that has recently been found to be frequently mutated in patients with endometriosis, endometrial cancer and ovarian cancer. Here, we present the first computational analysis of 13 somatic missense PTEN mutations associated with these phenotypes. We found that a majority of the mutations are associated in conserved positions within the active site and are clustered within the signature motif, which contain residues that play a crucial role in loop conformation and are essential for catalysis. In silico analyses were utilized to identify the putative effects of these mutations. In addition, coarse‐grained models of both wild‐type (WT) PTEN and mutants were constructed using elastic network models to explore the interplay of the structural and global dynamic effects that the mutations have on the relationship between genotype and phenotype. The effects of the mutations reveal that the local structure and interactions affect polarity, protein structure stability, electrostatic surface potential, and global dynamics of the protein. Our results offer new insight into the role in which PTEN missense mutations contribute to the molecular mechanism and genotypic‐phenotypic correlation of endometriosis, endometrial cancer, and ovarian cancer. Proteins 2016; 84:1625–1643. © 2016 Wiley Periodicals, Inc.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Photomorphogenesis of the root system in developing sunflower seedlings: a role for sucrose

• The domestic sunflower (Helianthus annuus L. cv. ‘Giganteus’) has been used since the 19th century as a model plant for the study of seedling development in darkness and white light (WL) (scoto‐ versus photomorphogenesis). However, most pertinent studies have focused on the developmental patterns of the hypocotyl and cotyledons, whereas the root system has been largely ignored.
• In this study, we analysed entire sunflower seedlings (root and shoot) and quantified organ development in the above‐ and belowground parts of the organism under natural (non‐sterile) conditions.
• We document that seedlings, raised in moist vermiculite, are covered with methylobacteria, microbes that are known to promote root development in Arabidopsis. Quantitative data revealed that during photomorphogenesis in WL, the root system expands by 90%, whereas stem elongation is inhibited, and hook opening/cotyledon expansion occurs. Root morphogenesis may be mediated via imported sucrose provided by the green, photosynthetically active cotyledons. This hypothesis is supported by the documented effect of sucrose on the induction of lateral root initials in sunflower cuttings. Under these experimental conditions, phytohormones (auxin, cytokinin, brassinolide) exerted little effect on root and cotyledon expansion, and no hormone‐induced initiation of lateral roots was observed.
• It is concluded that sucrose not only acts as an energy source to fuel cell metabolism but is also a shoot‐derived signalling molecule that triggers root morphogenesis.

     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

Chemistry of the consumption and excretion of the bumphead parrotfish (Bolbometopon muricatum), a coral reef mega-consumer

Coral Reefs - Bolbometopon muricatum are ecologically unique mega-consumers in coral reef ecosystems. They primarily divide their dietary intake between living scleractinian corals and coral rock,...     

Bicaudal‐D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule‐based motors is essential for cell organisation and function. The Bicaudal‐D (BicD) protein participates in the transport of a subset of cargoes by the minus‐end‐directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co‐precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin‐mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high‐frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin‐associated trafficking processes and show a novel requirement for microtubule‐based motor transport in the synaptic vesicle cycle.     

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.     

Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.     

Evaluation of ivermectin for treatment of hair loss syndrome in black-tailed deer

Since 1997, numerous Columbian black-tailed deer (Odocoileus hemionus columbianus) in western Washington (USA) have developed a hair loss syndrome that often preceded emaciation, debilitation, pneumonia, and death. To study this syndrome, eight affected free-ranging Columbian black-tailed deer fawns were captured from western Washington in February 1999 to determine the effect of ivermectin treatment. Fecal examinations indicated that the internal parasites were Dictyocaulus viviparus, Parelaphostrongylus sp., Trichuris sp., Moniezia sp., Eimeria spp., and gastrointestinal strongyles. Biting lice (Tricholipeurus parallelus) were observed on all deer, with up to 5 lice/cm(2) on the index areas counted. Three deer were treated with ivermectin subcutaneously at doses between 0.2 and 1.3 mg/kg of body weight monthly for four consecutive months, and five control deer received no anthelmintic treatment. Complete blood counts, parasite evaluations, weight gains, and hair loss evaluations were used to assess effectiveness of treatment. Two untreated deer died during the experiment compared with no deaths among the three treated deer. Treated deer gained significantly more weight (P<0.05) than the untreated deer (22.4 vs. 12.6 kg, respectively) that survived the experiment, had significantly fewer parasite eggs and larvae (P<0.05) in feces and significantly fewer nematodes (P<0.05) at necropsy, and regrew their hair at a faster rate than untreated deer. Lice and all nematode eggs and larval stages in feces were eliminated or greatly reduced following treatment. On the basis of these data, excessive louse populations, gastrointestinal nematodes, and the lung-worms Parelaphostrongylus sp. and D. viviparus, might be important predisposing factors for this hair loss condition and death of affected animals.