Pathway engineering by designed divergent evolution

Designed divergent evolution is a proposed protein engineering methodology to redesign enzyme function. The methodology was developed on the basis of the theories of divergent molecular evolution: (i) enzymes with more active and specialized functions have evolved from ones with promiscuous functions; (ii) this process is driven by small numbers of amino acid substitutions (plasticity); and (iii) the effects of double or multiple mutations are often additive (quasi-additive assumption). Thus, in many cases the impact of multiple mutations can be calculated by first determining the effects of a mutation at a single position and subsequently summing these effects using the quasi-additive assumption. In this way, the shape of the fitness landscape of a particular enzyme function can be estimated. The combinations of mutations predicted to yield global optima for desired functions can then be selected and introduced into the enzymes. The methodology has been demonstrated to be very powerful to redesign enzyme function. The use of multiple redesigned enzymes in novel or reconstructed metabolic pathways will enable the production of natural and unnatural products that will find use as pharmaceuticals, agrochemicals and many other applications.     

Flower form alteration by genetic transformation with the class B MADS-box genes of <Emphasis Type="Italic">Agapanthus</Emphasis><Emphasis Type="Italic">praecox</Emphasis> spp. <Emphasis Type="Italic">orientalis</Emphasis> in transgenic dicot and monocot plants

The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEF- and GLO-like genes, and the B-function is provided by heterodimers of a DEF- and a GLO-like gene product. In the present study, dicot (tobacco and lettuce) and monocot (Tricyrtis hirta) plants were transformed with the GLO-like gene of Agapanthus praecox ssp. orientalis ApGLO alone or in combination with the DEF-like gene of the same plant ApDEF. In two out of 10 transgenic tobacco plants containing ApGLO, sepals partially converted into petaloid organs. For lettuce, ray florets of four out of nine transgenic plants containing ApGLO also developed additional petaloid organs. In two out of five transgenic T. hirta plants containing both ApGLO and ApDEF, organs developed in whorl 4 showed noticeable morphological alteration: they were much longer compared with carpels of non-transgenic plants, and had purple spots overall on the surface as filaments of non-transgenic plants. No morphological alterations were observed in vegetative organs between transgenic and non-transgenic plants for all the three species. The results obtained in the present study indicate a possibility of molecular breeding for flower form alteration by genetic transformation with the class B MADS-box gene(s) of heterologous plant species.     

superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice

Cleistogamy is an efficient strategy for preventing gene flow from genetically modified (GM) crops. We identified a cleistogamous mutant of rice harbouring a missense mutation (the 45th residue isoleucine to threonine; I45T) in the class-B MADS-box gene SUPERWOMAN1 ( SPW1 ), which specifies the identities of lodicules (equivalent to petals) and stamens. In the mutant, spw1-cls , the stamens are normal, but the lodicules are transformed homeotically to lodicule–glume mosaic organs, thereby engendering cleistogamy. Since this mutation does not affect other agronomic traits, it can be used in crosses to produce transgenic lines that do not cause environmental perturbation. Molecular analysis revealed that the reduced heterodimerization ability of SPW1^I45T with its counterpart class-B proteins OsMADS2 and OsMADS4 caused altered lodicule identity. spw1-cls is the first useful mutant for practical gene containment in GM rice. Cleistogamy is possible in many cereals by engineering class-B floral homeotic genes and thereby inducing lodicule identity changes.     

Macroautophagy is essential for killing of intracellular Burkholderia pseudomallei in human neutrophils

Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.     

Regulation of Autophagy and Its Associated Cell Death by “Sphingolipid Rheostat”: RECIPROCAL ROLE OF CERAMIDE AND SPHINGOSINE 1-PHOSPHATE IN THE MAMMALIAN TARGET OF RAPAMYCIN PATHWAY*

The role of “sphingolipid rheostat” by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(−)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(−) or C₂-ceramide. AA(−) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(−). S1P exerts biological actions via cell surface receptors, and S1P₃ among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P₃ in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(−) or C₂-ceramide. Whereas S1P treatment of S1P₃ overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(−) or C₂-ceramide. These results indicate that S1P-S1P₃ plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(−)- or C₂-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P₃ engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P₃ signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.     

Motor Neuron-specific Disruption of Proteasomes,but Not Autophagy,Replicates Amyotrophic Lateral Sclerosis

Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.     

In vivo determination of the Achilles tendon moment arm in three-dimensions

Two-dimensional methods have been applied to determine the Achilles tendon moment arm in previous studies, although the talocrural joint rotates in three-dimension. The purpose of this study was to develop a method for determining the Achilles tendon moment arm in three-dimensions (3DMA). A series of sagittal ankle images were obtained at ankle positions of -20°, -10° (dorsiflexed position), 0° (neutral position), +10°, +20°, and +30° (plantarflexed position). The talocrural joint axis was determined as the finite helical axis of the ankle joint over 20° of displacement, and the 3DMA was determined as the shortest distance from the talocrural joint axis to the line of action of the Achilles tendon force. The corresponding 2DMA was determined with the center of rotation method using the images captured on the sagittal plane passing through the mid-point of the medio-lateral width of the tibia. The 3DMA ranged from 35 to 41 mm across various ankle positions and was, on average, 11 mm smaller than 2DMA. The difference between the two measures was attributable primarily to the deviations of the talocrural joint axis from the anatomical medio-lateral direction. The deviations on the coronal plane (21.4±20.7°) and on the transverse planes (14.8±22.6°) accounted for the errors of 1.3 mm and 3.0 mm, respectively. In addition, selecting either a medially or laterally misaligned sagittal-plane image for determining the 2DMA gave rise to error by 3.5 mm. The remaining difference was accounted for by the random measurement error.     

Amelioration of collagen-induced arthritis by a novel S1P1 antagonist with immunomodulatory activities

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.     

Glycosylation of Simian Immunodeficiency Virus Influences Immune-Tissue Targeting during Primary Infection, Leading to Immunodeficiency or Viral Control

Glycans of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) play pivotal roles in modulating virus-target cell interactions. We have previously reported that, whereas SIVmac239 is pathogenic, its deglycosylated essentially nonpathogenic mutant (Δ5G) serves as a live-attenuated vaccine, although both replicate similarly during primary infection. These findings prompted us to determine whether such a polarized clinical outcome was due to differences in the immune tissues targeted by these viruses, where functionally and phenotypically different memory CD4(+) T cells reside. The results showed that Δ5G replicates in secondary lymphoid tissue (SLT) at 1- to 2-log-lower levels than SIVmac239, whereas SIVmac239-infected but not Δ5G-infected animals deplete CXCR3(+) CCR5(+) transitional memory (TrM) CD4(+) T cells. An early robust Δ5G replication was localized to small intestinal tissue, especially the lamina propria (effector site) rather than isolated lymphoid follicles (inductive site) and was associated with the induction and depletion of CCR6(+) CXCR3(-) CCR5(+) effector memory CD4(+) T cells. These results suggest that differential glycosylation of Env dictates the type of tissue-resident CD4(+) T cells that are targeted, which leads to pathogenic infection of TrM-Th1 cells in SLT and nonpathogenic infection of Th17 cells in the small intestine, respectively.     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.