Rate-limiting Pathway Research Articles

HY5 and PIF antagonistically regulate HMGR expression and sterol biosynthesis in Arabidopsis thaliana

Secondary metabolites play multiple crucial roles in plants by modulating various regulatory networks. The biosynthesis of these compounds is unique to each species and is intricately controlled by a range of developmental and environmental factors. While light's role in certain secondary metabolites is evident, its impact on sterol biosynthesis remains unclear. Previous studies indicate that ELONGATED HYPOCOTYL5 (HY5), a bZIP transcription factor, is pivotal in skotomorphogenesis to photomorphogenesis transition. Additionally, PHYTOCHROME INTERACTING FACTORs (PIFs), bHLH transcription factors, act as negative regulators. To unveil the light-dependent regulation of the mevalonic acid (MVA) pathway, a precursor for sterol biosynthesis, mutants of light signaling components, specifically hy5–215 and the pifq quadruple mutant (pif 1,3,4, and 5), were analyzed in Arabidopsis thaliana. Gene expression analysis in wild-type and mutants implicates HY5 and PIFs in regulating sterol biosynthesis genes. DNA-protein interaction analysis confirms their interaction with key genes like AtHMGR2 in the rate-limiting pathway. Results strongly suggest HY5 and PIFs' pivotal role in light-dependent MVA pathway regulation, including the sterol biosynthetic branch, in Arabidopsis, highlighting a diverse array of light signaling components finely tuning crucial growth pathways.

De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae

Plant styrylpyrones exerting well-established neuroprotective properties have attracted increasing attention in recent years. The ability to synthesize each individual styrylpyrone in engineered microorganisms is important to understanding the biological activity of medicinal plants and the complex mixtures they produce. Microbial biomanufacturing of diverse plant-derived styrylpyrones also provides a sustainable and efficient approach for the production of valuable plant styrylpyrones as daily supplements or potential drugs complementary to the prevalent agriculture-based approach. In this study, we firstly demonstrated the heterogenous biosynthesis of two 7,8-saturated styrylpyrones (7,8-dihydro-5,6-dehydrokavain (DDK) and 7,8-dihydroyangonin (DHY)) and two 7,8-unsaturated styrylpyrones (desmethoxyyangonin (DMY) and yangonin (Y)), in Saccharomyces cerevisiae. Although plant styrylpyrone biosynthetic pathways have not been fully elucidated, we functionally reconstructed the recently discovered kava styrylpyrone biosynthetic pathway that has high substrate promiscuity in yeast, and combined it with upstream hydroxycinnamic acid biosynthetic pathways to produce diverse plant-derived styrylpyrones without the native plant enzymes. We optimized the de novo pathways by engineering yeast endogenous aromatic amino acid metabolism and endogenous double bond reductases and by CRISPR-mediated δ-integration to overexpress the rate-limiting pathway genes. These combinatorial engineering efforts led to the first three yeast strains that can produce diverse plant-derived styrylpyrones de novo, with the titers of DDK, DMY and Y at 4.40 μM, 1.28 μM and 0.10 μM, respectively. This work has laid the foundation for larger-scale styrylpyrone biomanufacturing and the complete biosynthesis of more complicated plant styrylpyrones.

Molecular targets and approaches to restore autophagy and lysosomal capacity in neurodegenerative disorders

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity.

Distinct Expression of the Two NO-Forming Nitrite Reductases in Thermus antranikianii DSM 12462T Improved Environmental Adaptability

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2-) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Chemical characterization and anaerobic biodegradability of hydrothermal liquefaction aqueous products from mixed-culture wastewater algae

This study examined the chemical characteristics and the anaerobic degradability of the aqueous product from hydrothermal liquefaction (HTL-ap) from the conversion of mixed-culture algal biomass grown in a wastewater treatment system. The effects of the HTL reaction times from 0 to 1.5h, and reaction temperatures from 260°C to 320°C on the anaerobic degradability of the HTL-ap were quantified using biomethane potential assays. Comparing chemical oxygen demand data for HTL-ap from different operating conditions, indicated that organic matter may partition from organic phase to aqueous phase at 320°C. Moderate lag phase and the highest cumulative methane production were observed when HTL-ap was obtained at 320°C. The longest lag phase and the smallest production rate were observed in the process fed with HTL-ap obtained at 300°C. Nevertheless, after overcoming adaptation issues, this HTL-ap led to the second highest accumulated specific methane production. Acetogenesis was identified as a possible rate-limiting pathway.

Body composition determines direct FFA storage pattern in overweight women

Direct FFA storage in adipose tissue is a recently appreciated pathway for postabsorptive lipid storage. We evaluated the effect of body fat distribution on direct FFA storage in women with different obesity phenotypes. Twenty-eight women [10 upper body overweight/obese (UBO; WHR >0.85, BMI >28 kg/m(2)), 11 lower body overweight/obese (LBO; WHR <0.80, BMI >28 kg/m(2)), and 7 lean (BMI <25 kg/m(2))] received an intravenous bolus dose of [9,10-(3)H]palmitate- and [1-(14)C]triolein-labeled VLDL tracer followed by upper body subcutaneous (UBSQ) and lower body subcutaneous (LBSQ) fat biopsies. Regional fat mass was assessed by combining DEXA and CT scanning. We report greater fractional storage of FFA in UBSQ fat in UBO women compared with lean women (P < 0.01). The LBO women had greater storage per 10(6) fat cells in LBSQ adipocytes compared with UBSQ adipocytes (P = 0.04), whereas the other groups had comparable storage in UBSQ and LBSQ adipocytes. Fractional FFA storage was significantly associated with fractional VLDL-TG storage in both UBSQ (P < 0.01) and LBSQ (P = 0.03) adipose tissue. In conclusion, UBO women store a greater proportion of FFA in the UBSQ depot compared with lean women. In addition, LBO women store FFA more efficiently in LBSQ fat cells compared with UBSQ fat cells, which may play a role in development of their LBO phenotype. Finally, direct FFA storage and VLDL-TG fatty acid storage are correlated, indicating they may share a common rate-limiting pathway for fatty acid storage in adipose tissue.

Prostate specific antigen reduction following statin therapy: Mechanism of action and review of the literature

Prostate specific antigen (PSA) is a serine protease that is exclusively produced in the prostate, and its detection is the only laboratory test available for screening men for prostate cancer (PC). The interpretation of the assay is difficult since it is specific for prostate tissue and cellular growth, but not for PC. Pharmacologic therapy for hyperlipidemia, such as statins, may influence prostate cellular growth and subsequently PSA levels in patients. Dysregulated cellular growth in the prostate is mediated by inhibiting the rate-limiting pathway step in cholesterol synthesis, thereby decreasing isoprenylate intermediates, decreasing cholesterol rich cellular membrane domains, and down-regulating androgen and estrogen receptors. Statins, with variable efficacy, have been previously shown to inhibit cellular inflammation, angiogenesis, proliferation, migration/adhesion, and invasion, while promoting apoptosis in prostate cells by inhibiting the conversion of HMG-CoA to mevalonate. An individual statin's molecular structure, need for enzymatic conversion, bioavailability, and peripheral tissue concentration may partially account for differing properties. By inhibiting prostatic cellular growth and promoting apoptosis, statins may subsequently decrease PSA levels, an effect recently observed in cohorts. There is scientific and clinical evidence supporting the observations that statins are associated with an overall reduction in serum PSA in men, when used for greater than 6 months, and especially when used for greater than 2 years.

An evaluation of plasma homocysteine in the assessment of vitamin B12 status of pasture-fed sheep

AIM: To assess the diagnostic potential of concentrations of homocysteine (Hcy) in plasma in relation to those of methylmalonic acid (MMA) and vitamin B12, as predictors of responsiveness of young sheep to supplementation with vitamin B12. METHODS: Eighty-two ewes grazing ryegrass-white clover pasture were used, 39 of which had been supplemented with a Co bullet and 43 unsupplemented. Thirty days after commencement of parturition their lambs (n=53 and 59, respectively) were randomly allocated into one of two treatments, in a 2 × 2 factorial design. Half of the lambs from each group of ewes received an injection of vitamin B12, while the remainder were controls. The trial commenced 31 October 2001 (Day 0), and continued until 01 May 2002 (Day 182). All lambs were weighed, and blood samples taken from 16 identified animals from each treatment group, at approximately monthly intervals. Changes in concentrations of Hcy, vitamin B12 and MMA in plasma, and liveweight gain (LWG) of the treatment groups were evaluated during the suckling (Days 0–89) and post-weaning (Days 90–182) periods. RESULTS: Mean LWG was 40% greater in supplemented than unsupplemented lambs. The concentrations of vitamin B12 and MMA in plasma in the unsupplemented lambs were in the deficient reference ranges of <170 pmol/L and >16 µmol/L, respectively. Mean monthly concentrations of Hcy in plasma ranged from 1.5 to 4.5 µmol/L but showed no pattern of response to vitamin B12 deficiency or supplementation. CONCLUSIONS: Measurement of the concentration of Hcy in plasma as a metabolic indicator of reduced methylation capability of sheep on typical pastures in New Zealand appeared to have little value in detection of vitamin B12 responsiveness, and was less sensitive than the concentration of the vitamin itself or the indicator of adenosyl-cobalamin deficiency, MMA, in plasma. The possibility that concentrations of Hcy in plasma remain low due to re-methylation of Hcy to methionine via the alternative betaine-choline rather than the vitamin B12-dependent methyl-tetrahydrofolate metabolic pathway is rejected, but the possibility is raised that high rates of trans-sulphuration of Hcy to cysteine in the gastrointestinal tract of grazing sheep could be responsible. CLINICAL RELEVANCE: The propionate-succinate pathway appears to be the first rate-limiting pathway in vitamin B12 deficiency, and the product of disruption of this pathway, increased MMA, is the most reliable indicator of metabolic abnormality in predicting responsiveness to supplementation.

A simple algebraic cancer equation: calculating how cancers may arise with normal mutation rates

BackgroundThe purpose of this article is to present a relatively easy to understand cancer model where transformation occurs when the first cell, among many at risk within a colon, accumulates a set of driver mutations. The analysis of this model yields a simple algebraic equation, which takes as inputs the number of stem cells, mutation and division rates, and the number of driver mutations, and makes predictions about cancer epidemiology.MethodsThe equation [p = 1 - (1 - (1 - (1 - u)d)k)Nm ] calculates the probability of cancer (p) and contains five parameters: the number of divisions (d), the number of stem cells (N × m), the number of critical rate-limiting pathway driver mutations (k), and the mutation rate (u). In this model progression to cancer "starts" at conception and mutations accumulate with cell division. Transformation occurs when a critical number of rate-limiting pathway mutations first accumulates within a single stem cell.ResultsWhen applied to several colorectal cancer data sets, parameter values consistent with crypt stem cell biology and normal mutation rates were able to match the increase in cancer with aging, and the mutation frequencies found in cancer genomes. The equation can help explain how cancer risks may vary with age, height, germline mutations, and aspirin use. APC mutations may shorten pathways to cancer by effectively increasing the numbers of stem cells at risk.ConclusionsThe equation illustrates that age-related increases in cancer frequencies may result from relatively normal division and mutation rates. Although this equation does not encompass all of the known complexity of cancer, it may be useful, especially in a teaching setting, to help illustrate relationships between small and large cancer features.

Small Molecule RPE65 Antagonists Limit the Visual Cycle and Prevent Lipofuscin Formation

The accumulation of the lipofuscin fluorophores in retinal pigment epithelial (RPE) cells leads to the blinding degeneration characteristic of Stargardt disease and related forms of macular degeneration. RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cycle. Inhibiting visual cycle function with small molecules is required to prevent the formation of the retinotoxic lipofuscins. This in turn requires identification of rate-limiting steps in the operation of the visual cycle. Specific, non-retinoid isoprenoid compounds are described here, and shown through in both in vitro and in vivo experiments, to serve as antagonists of RPE65, a protein that is essential for the operation of the visual cycle. These RPE65 antagonists block regeneration of 11-cis-retinal, the chromophore of rhodopsin, thereby demonstrating that RPE65 is at least partly rate-limiting in the visual cycle. Furthermore, chronic treatment of a mouse model of Stargardt disease with the RPE65 antagonists abolishes the formation of A2E. Thus, RPE65 is also on the rate-limiting pathway to A2E formation. These nontoxic isoprenoid RPE65 antagonists are candidates for the treatment of forms of macular degeneration wherein lipofuscin accumulation is an important risk factor. These antagonists will also be used to probe the molecular function of RPE65 in vision.

Specificity of Binding of all-trans-Retinyl Ester to RPE65

Membrane-bound RPE65 (mRPE65) is a binding protein for all-trans-retinyl esters, which are the substrates for the isomerization reaction that completes the visual cycle. RPE65 is essential for rhodopsin regeneration and, hence, for vision. As RPE65 appears to be part of the rate-limiting pathway in the visual cycle, specific antagonists of the molecule will be important in evaluating its full physiological role. The protein is known to stereoselectively bind all-trans-retinyl esters (tREs), with dissociation constants in the 50 nM range. This study explores the overall binding specificity of RPE65 with respect to both retinoids and other isoprenoids in an effort to define the specificity of binding, and to begin the process of designing specific antagonists for it. The nature of the specificity directed toward the three main structural elements (retinoid, linker, and acyl moieties) in the tRE molecule is reported. In the all-trans-retinyl ester series, binding affinity increased as a function of the hydrophobicity of the fatty acyl group. In the linker region, binding affinities were little affected by amide, ketone, and ether replacements for the carboxy ester moiety of the naturally occurring tRE ligand. Finally, modifications in the all-trans-retinoid moiety are also tolerated. For example, E,E-farnesyl palmitate binds with approximately the same affinity as does all-trans-retinyl palmitate. Other isoprenoid analogues also bind, as do truncated retinoids in the beta-ionone series. Therefore, mRPE65 is a moderately specific retinoid binding protein directed at long chain all-trans-retinyl esters.

Effects of oxygen on kynurenine-3-monooxygenase activity

Kynurenine-3-monooxygenase (KM), the third enzyme in the kynurenine (KYN) pathway from tryptophan to quinolinic acid (QA), is a monooxygenase requiring oxygen, NADPH and FAD for the catalytic oxidation of L-kynurenine to 3-hydroxykynurenine and water. KM is innately low in the brain and similar in activity to indoleamine oxidase, the rate-limiting pathway enzyme. Accumulation in the CNS of QA, a known excitotoxin, is proposed to cause convulsions in several pathologies. Thus, we theorized that hyperbaric oxygen (HBO) induced convulsions arise from increased QA via oxygen Km effects on this pathway [Brown OR, Draczynska-Lusiak. Oxygen activation and inactivation of quinolinate-producing and iron-requiring 3-hydroxyanthranilic acid oxidase: a role in hyperbaric oxygen-induced convulsions? Redox Report 1995; 1: 383–385]. To complement prior studies on the effects of oxygen on pathway enzymes, in this paper we report the effects of oxygen on KM. Brain and liver KM enzyme are not known to be identical, and some systemically-produced KYN pathway intermediates can permeate the brain and might stimulate the brain pathway. Thus, KM from both brain and liver was assayed at various oxygen substrate concentrations to evaluate, in vitro, the potential effects of increases in oxygen, as would occur in mammals breathing therapeutic and convulsive HBO. In crude tissue extracts, KM was not activated during incubation in HBO up to 6 atm. The effects of oxygen as substrate on brain and liver KM activity was nearly identical: activity was nil at zero oxygen with an apparent oxygen Km of 20–22 µM. Maximum KM activity occurred at about 1000 µM oxygen and decreased slightly to plateau from 2000 to 8000 µM oxygen. This compares to approximately 30–40 µM oxygen typically reported for brain tissue of humans or rats breathing air, and an unknown but surely much lower value (perhaps below 1 µM) intracellularly at the site of KM. Thus HBO, as used therapeutically and at convulsive pressures, likely stimulates flux through the KM-catalyzed step of the KYN pathway in liver and in brain and could increase brain QA, by Km effects on brain KM, or via increased KM pathway intermediates produced systemically (in liver) and transported into the brain.

A new method for examining the complexity and relationships of “timers” in developing systems

Simple methods are developed for analyzing the rate-limiting pathways, or “developmental timers,” for consecutive stages in a developing system. Two conditions are first defined for short and long timing to a developmental stage. Shifts are then performed at time intervals from short to long and long to short conditions. The total time to the stage (time under first condition plus time under second condition) is scored and plotted as a function of the time of shift, resulting in two plots, one for shifts from the short to long condition, and the other for shifts from the long to short condition. Each plot is then analyzed for the number of components, slopes of components, absolute times of origins and termini of components, and discontinuities between components. This information is then used (1) to distinguish between single- and multiple-component timers, (2) to assess the sensitivity of each timer component to the change in the environmental condition employed in the method, including reversibility, (3) to test for the addition of a new timer component under long conditions, and (4) to test for an identity change of a timer component between short and long conditions. These interpretations in turn provide a minimum estimate of the complexity of the rate-limiting pathway to a developmental stage, temporally define major transition points between timer components, and provide some insight into the nature of timer components. By characterizing the rate-limiting pathway from the origin of a developmental program for each consecutive stage in that program, distinctions can also be made between single, parallel, sequential, and branching timer relationships. From these interpretations, a detailed temporal “map” of the rate-limiting program can be generated for any developmental system in which consecutive stages can be reproducibly monitored with time.

Differentiation and dedifferentiation can function simultaneously and independently in the same cells in Dictyostelium discoideum

When developing cultures of Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, cells recapitulate the stages they had progressed through prior to disaggregation in a fraction of the original time. If developing cultures are disaggregated and the cells resuspended in nutrient medium, they retain this capacity for 1.5 hr and then synchronously and rapidly revert to the slow timing of log phase cells. Loss of the capacity to recapitulate morphogenesis rapidly is referred to as the “erasure event.” Following the erasure event, cells systematically lose developmentally acquired functions in a defined temporal sequence of dedifferentiation. Cells which have just passed through the erasure event can be stimulated to reenter the developmental program, even though they still possess several aggregation-associated functions acquired during the initial developmental program. In this report, we have tested whether cells stimulated to reenter the developmental program immediately after the erasure event progress along the same rate-limiting pathway leading to aggregation as they did during initial development and whether this rate-limiting pathway can run simultaneously with and independently of the sequence of dedifferentiation. Results are presented which demonstrate (1) that the erasure event resets the rate-limiting pathway for development back to zero and that erased cells reentering development progress along the same rate-limiting pathway as naive log phase cells, (2) that the loss of an aggregation-associated function late in the sequence of dedifferentiation is completely blocked by the addition of cycloheximide, but not cAMP, just prior to the expected time of loss, and (3) that differentiation and dedifferentiation can function simultaneously and independently in the same cells, even though the former leads to the acquisition and the latter to the loss of the same aggregation-associated functions (in this case EDTA-resistant adhesion and cAMP-stimulated motility).