Plasma Membrane Level Research Articles

Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions.

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region—the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region—regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ΔRE and ΔRI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ΔRI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22.

Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.

AOP1, a New Live Cell Assay for the Direct and Quantitative Measure of Intracellular Antioxidant Effects.

Taking advantage of Light Up Cell System (LUCS) technology, which allows for fine monitoring of reactive oxygen species (ROS) production inside live cells, a new assay called Anti Oxidant Power 1 (AOP1) was developed to specifically measure ROS and/or free-radical scavenging effects inside living cells. This method is quantitative and EC50s obtained from AOP1 dose-response experiments were determined in order to classify the intracellular antioxidant efficacy of 15 well known antioxidant compounds with different hydrophilic properties. Six of them (epigallocatechin gallate, quercetin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethoxyquin, resveratrol) gave EC50s in the range of 7–64 μM, four (Trolox, catechin, epicatechin, EUK134) in the range of 0.14 to 1 mM, and 5 (sulforaphane, astaxanthin, α- and γ-tocopherols, vitamin E acetate) showed only partial or no effect. Interestingly, effects with measurable EC50s were observed for compounds with hydrophilic properties (LogP ≤ 5.3), while all antioxidants known to act at the plasma membrane level (LogP ≥ 10.3) had partial or no effect. Sulforaphane, a hydrophilic but strict Keap1/Nrf2 pathway enhancer, did not show any effect either. Importantly, AOP1 assay captures both antioxidant and prooxidant effects. Taken together, these results led us to the conclusion that AOP1 assay measures antioxidant effect of compounds that selectively enter the cell, and act as free radical scavengers in the cytosol and/or nucleus level.

Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories

Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoCH419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoCH419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.

Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport.

Focus on Triple-Negative Breast Cancer: Potassium Channel Expression and Clinical Correlates.

Despite improvements in early diagnosis and treatment, breast cancer is still a major health problem worldwide. Among breast cancer subtypes, the most challenging and harder to treat is represented by triple-negative molecular subtype. Due to its intrinsic features this subtype cannot be treated neither with hormonal therapy (since it does not express estrogen or progesterone receptors) nor with epidermal growth factor receptor 2 (HER2) inhibitors (as it does not express high levels of this protein). For these reasons, the standard of care for these patients is represented by a combination of surgery, radiation therapy and chemotherapy. In this scenario, searching for novel biomarkers that might help both in diagnosis and therapy is mandatory. In the last years, it was shown that different families of potassium channels are overexpressed in primary breast cancers. The altered ion channel expression may be useful for diagnostic and therapeutic purposes due to some peculiar characteristics of this class of molecules. Ion channels are defined as pore-forming transmembrane proteins regulating passive ion fluxes in the cells. Ion channels represent good potential markers since, being localized at the plasma membrane level, their detection and block with specific drugs and antibodies might be fast and tunable. This review focuses on triple-negative breast cancers and recapitulates the current knowledge about potassium channels' clinical relevance and their potential use in the clinical setting, for triple-negative breast cancer diagnosis and therapy.

HIV-1 Tat \u2013 TLR4/MD2 interaction drives the expression of IDO-1 in monocytes derived dendritic cells through NF-\u03baB dependent pathway

In the present study we showed that HIV-1 Tat protein stimulated the expression of Indoleamine 2,3 dioxygenase (IDO) -1 in human monocytes derived dendritic cells (MoDC) but not IDO-2 by acting directly at the cell membrane level. This induction of IDO-1 is dependent on the secondary structure of Tat protein, since stimulation with a chemically oxidized Tat protein loses its capacity to induce the production of IDO-1. Among the variety of candidate receptors described for Tat, we demonstrated that Tat protein interacted physically with TLR4/MD2 complex. Strikingly, blockade of Tat-TLR4 interaction by anti-TLR4 antibodies (clone HTA125), LPS-RS, a known TLR4 antagonist, or by soluble recombinant TLR4/MD2 complex inhibited strongly or totally the capacity of Tat to induce IDO-1 in MoDC while such treatments had no effect on IFN-γ-induced IDO-1. Furthermore, we showed that the activation of the transcription factor NF-κB by Tat is essential for the production of IDO-1 by human MoDC. Indeed, Tat activated NF-κB pathway in MoDC as demonstrated by the phosphorylation of p65 in Tat-treated MoDC. Further, we demonstrate that the stimulation of IDO-1 by Tat or by IFN-γ was totally or partially inhibited in the presence of NF-κB inhibitor respectively. These results suggest that Tat and IFN-γ act probably by two distinct mechanisms to induce the production of IDO-1. Our results clearly demonstrated that, although TLR4 pathway is necessary for Tat-induced IDO-1 in MoDC, it seems not to be sufficient since stable transfection of a functional TLR4/MD2 pathway in HEK or HeLa cell lines which are endogenously defectives for TLR4, did not restore the capacity of Tat to induce IDO-1 while IFN-γ treatment induces IDO-1 in HeLa cells independently of TLR4 pathway. These results suggest the involvement of additional stimuli in addition to TLR4 pathway which remain to be identified. Altogether our results demonstrated that, in human MoDC, HIV-1 Tat protein induced IDO-1 expression and activity in a NF-κB dependent-manner by recruiting TLR4 pathway.

Synergistic antiviral effect of hydroxychloroquine and azithromycin in combination against SARS-CoV-2: What molecular dynamics studies of virus-host interactions reveal

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.

Morphological and structural response of Bacillus sp. SFC 500‐1E after Cr(VI) and phenol treatment

Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.

Regulation of Vessel Permeability by TRP Channels.

The vascular endothelium constitutes a semi-permeable barrier between blood and interstitial fluids. Since an augmented endothelial permeability is often associated to pathological states, understanding the molecular basis for its regulation is a crucial biomedical and clinical challenge. This review focuses on the processes controlling paracellular permeability that is the permeation of fluids between adjacent endothelial cells (ECs). Cytosolic calcium changes are often detected as early events preceding the alteration of the endothelial barrier (EB) function. For this reason, great interest has been devoted in the last decades to unveil the molecular mechanisms underlying calcium fluxes and their functional relationship with vessel permeability. Beyond the dicotomic classification between store-dependent and independent calcium entry at the plasma membrane level, the search for the molecular components of the related calcium-permeable channels revealed a difficult task for intrinsic and technical limitations. The contribution of redundant channel-forming proteins including members of TRP superfamily and Orai1, together with the very complex intracellular modulatory pathways, displays a huge variability among tissues and along the vascular tree. Moreover, calcium-independent events could significantly concur to the regulation of vascular permeability in an intricate and fascinating multifactorial framework.

Abstract IA08: Lipid dependence of KRAS plasma membrane interactions

Abstract RAS proteins must localize to the plasma membrane (PM) in order to function. We have examined how different RAS PM anchors select and assemble highly specific phospholipids into signaling platforms called nanoclusters, how these transient lipid assemblies impact signal transmission, and in consequence how the lipid composition of the PM can operate as high-level regulator of RAS signal transmission. For example, the KRAS polybasic domain (PBD)-farnesyl membrane anchor exhibits exquisite specificity for phosphatidylserine (PtdSer) over other anionic phospholipids by virtue of defined structural dynamics of the C-terminal polybasic domain and farnesyl group. This specificity extends to PtdSer with specific combinations of lipid side chains. PtdSer binding is therefore essential for stable KRAS PM binding and the spatial organization of KRAS into nanoclusters. These and complementary results with RAC1 highlight the critical role of specific anionic PM lipids in the biologic function of small GTPases. In this context for KRAS we have now shown that the PM levels and lateral availability of PtdSer are critically dependent on sphingomyelin metabolism such that multiple perturbations of sphingomyelin content or distribution, or endolysosmal lipid trafficking, profoundly affect KRAS plasma membrane localization, nanoclustering, and function. Concordantly, directly targeting the molecular machinery that maintains PtdSer on the PM also abrogates KRAS function in multiple pancreatic cancer cell lines. These new observations suggest multiple new potential therapeutic targets to abrogate oncogenic KRAS signaling, which we are evaluating in model systems. Citation Format: John F. Hancock. Lipid dependence of KRAS plasma membrane interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA08.

Naloxone Is a Potential Binding Ligand and Activator of the Capsaicin Receptor TRPV1.

The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a μ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by [3H]-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.

Intake of Alpha-Linolenic Acid-Rich Perilla frutescens Leaf Powder Decreases Home Blood Pressure and Serum Oxidized Low-Density Lipoprotein in Japanese Adults.

Oxidized low-density lipoprotein (Ox-LDL) is known to be highly atherogenic. Thus, decreasing the blood levels of Ox-LDL through dietary means is an important approach to reduce cardiovascular events in high-risk individuals. In this randomized placebo-controlled human interventional trial, we aimed to evaluate whether Perilla frutescens leaf powder (PLP) ameliorates Ox-LDL and home blood pressure, along with its biological antioxidant potential. Healthy Japanese volunteers aged 30–60 years (n = 60) were randomized to PLP and placebo groups. The PLP group consumed PLP dried using a microwave under reduced pressure, and the placebo group consumed pectin fiber daily for 6 months. Home blood pressure, serum biochemical parameters, and fatty acid profiles of erythrocyte plasma membranes were analyzed. Plasma Ox-LDL levels significantly decreased in the PLP group but not in the placebo group. Mean changes in the biological antioxidant potential and alpha-linolenic acid levels in the erythrocyte plasma membrane were significantly increased in the PLP group than in the placebo group. In subjects with prehypertension (systolic blood pressure [SBP] ≥ 120 mmHg), the mean reduction in morning or nocturnal SBP was significantly greater in the PLP group than in the placebo group. Thus, PLP intake may be an effective intervention to prevent cardiovascular diseases.

Post-translational regulation of plasma membrane H+-ATPase is involved in the release of biological nitrification inhibitors from sorghum roots

It is an integral property of sorghum (Sorghum bicolor L.) to extensively release biological nitrification inhibitors (BNIs) under NH4+ nutrition, in comparison to NO3− nutrition. Our previous research indicated that plasma membrane (PM) H+-ATPase activity was stimulated by NH4+ and low rhizosphere pH, which in turn provided the driving force for BNIs release from sorghum roots. However, the regulatory mechanism of PM H+-ATPase itself in this regard is not fully elucidated. The present study thus aims at post-translational regulation of PM H+-ATPase via phosphorylation in response to NH4+ nutrition and its functional link to the release of BNIs from sorghum roots. A hydroponic system is used to grow sorghum with 1 mM NH4+ or NO3− as N source at pH 3.0 or pH 7.0 in root medium for the analysis of PM H+-ATPase and BNIs release. The effect of NH4+ on the regulation of PM H+-ATPase was further evaluated by the treatment of NO3−cultivated sorghum roots with different NH4+ concentrations (0.1~1 mM). In addition, fusicoccin (a stimulator of PM H+-ATPase) and vanadate (an inhibitor of PM H+-ATPase) were added to check the effect of PM H+-ATPase phosphorylation on BNIs release. Further, methionine sulphoximine (MSX), which inhibits glutamine synthetase, is used to analyze the effect of ammonium transport/assimilation process on the PM H+-ATPase and BNIs release. Microsomal membrane protein isolated from these roots was used for the test of PM H+-ATPase phosphorylation level by western blot technique. Meanwhile, the root exudates were collected for the analysis of BNIs. Higher amount of PM H+-ATPase protein with higher phosphorylation level were detected in sorghum roots in response to NH4+ and low rhizosphere pH, as compared to NO3− and high pH. Further, PM H+-ATPase protein amount and phosporylation level were dependent on the local supplement of NH4+ (from 0.1 ~ 1 mM) to roots. Nevertheless, the enhanced posphorylation level under all of these treatments was significantly higher than the enhanced protein level of PM H+ ATPase. Unlike protein level, phosphorylation level is closely correlated to the release of BNIs from sorghum roots. In addition, phosphorylation level of PM H+-ATPase adjusted by fusicoccin or vanadate directly affected the release of BNIs, irrespective of the protein level. In addition, ammonium assimilation inhibitor MSX caused decreased phosphorylation level of PM H+-ATPase without affecting the protein level, meanwhile inhibited the release of BNIs from sorghum roots. Our research suggests that phosphorylation of PM H+-ATPase is one of the important regulation mechanisms involved in the release of BNIs from sorghum roots. NH4+ stimulated PM H+-ATPase phosphorylation via excessive H+ generated by NH4+ assimilation in cytoplasm. The up regulation of PM H+-ATPase at post-translational level thus activated the H+ pumping activity to provide the driving force for BNIs release. A new hypothesis is proposed to elucidate the interplay of these functionally inter-linked processes involving ammonium-uptake, −assimilation, and H+-pumps activation in PM on the release of BNIs from sorghum roots.

PRESOWING STIMULATION OF ECHINACEA AND VALERIAN SEEDS WITH ULTRA-HIGH FREQUENCY ELECTROMAGNETIC FIELD

The article presents the results obtained by testing the previously proposed physical and mathematical model of the temperature distribution in the layer of poured seeds when irradiated with a high-frequency electromagnetic field. It has been proven that temperature is one of the factors influencing the consequences of pre-sowing seed treatment. We have found out that, along with heating, other physical processes occur in the seed structure at the level of cell membranes and membrane transport of substances. The transformation of protein structures, the polarization of cell membranes, and the intensification of metabolic processes under the influence of irradiation can lead to changes in the electrical properties of seeds and, as a consequence, can impact on the physiological state, germinability and growth energy of plants. The result of the seed exposure to high-frequency electromagnetic field depends on the electrical characteristics of the seeds. We have developed a technology for the pre-sowing treatment of Echinacea and Valerian seeds in order to obtain more ecologically pure raw materials for pharmacological industry. The study has demonstrated positive thermal and oscillation effects produced by the electromagnetic field on the structural elements of seeds. Based on the method of presowing stimulation of the seeds with a high-frequency electromagnetic field, the optimal modes for irradiation of Echinacea and Valerian seeds have been elaborated. It has been established that boosting the overall resistance of biological tissue, an increase in the polarization of cell membranes indicates an enhancement of metabolic processes and, as a result, stimulates the process of seed germination and rapid vegetation in the early stages of plant development. Theoretical calculations and experimental studies have evidenced positive results doe to implementation of the described technology.

The oligosaccharide portion of ganglioside GM1 regulates mitochondrial function in neuroblastoma cells.

The crucial role of ganglioside GM1 in the regulation of neural homeostasis has been assessed by several studies. Recently we shed new light on the molecular basis underlying GM1 effects demonstrating that GM1 oligosaccharide directly binds TrkA receptor and triggers MAPK pathway activation leading to neuronal differentiation and protection. Following its exogenous administration, proteomic analysis revealed an increased expression of proteins involved in several biochemical mechanisms, including mitochondrial bioenergetics. Based on these data, we investigated the possible effect of GM1 oligosaccharide administration on mitochondrial function. We show that wild-type Neuro2a cells exposed to GM1 oligosaccharide displayed an increased mitochondrial density and an enhanced mitochondrial activity together with reduced reactive oxygen species levels. Interestingly, using a Neuro2a model of mitochondrial dysfunction, we found an increased mitochondrial oxygen consumption rate as well as increased complex I and II activities upon GM1 oligosaccharide administration. Taken together, our data identify GM1 oligosaccharide as a mitochondrial regulator that by acting at the plasma membrane level triggers biochemical signaling pathway inducing mitochondriogenesis and increasing mitochondrial activity. Although further studies are necessary, the capability to enhance the function of impaired mitochondria points to the therapeutic potential of the GM1 oligosaccharide for the treatment of pathologies where these organelles are compromised, including Parkinson's disease.

Potassium alleviates ammonium toxicity in rice by reducing its uptake through activation of plasma membrane H+-ATPase to enhance proton extrusion

Potassium (K+) has been reported to alleviate ammonium (NH4+) toxicity in rice through some underlying mechanisms, but it still not clear. In addition, K+ is an important cation for activation of plasma membrane (PM) H+-ATPase activity. Here, we hypothesized that K+ alleviated NH4+ toxicity by mediating PM H+-ATPase function in rice root. In this study, rice plants were cultivated in hydroponic solution with various concentrations of K+ and NH4+. By concurrently supplying K+ with NH4+ or re-supplying K+ after NH4+ toxicity, we found that high K+ concentration reduced the NH4+ uptake rate, enhanced the H+ extrusion rate by the roots, and alleviated rice NH4+ toxicity. The gene expression levels of PM H+-ATPase members (OsA1, 3, 7, 8, and 9) were upregulated by application of increasing concentrations of K+ under NH4+ toxicity. The PM H+-ATPase activity and protein expression in rice roots were also enhanced. Furthermore, the enhancement of PM H+-ATPase activity by a specific stimulator (fusicoccin) rescued rice seedlings from NH4+ toxicity. Taken together, these results indicate that K+ can alleviate NH4+ toxicity, possibly by activating PM H+-ATPase to extrude more H+ and inhibit NH4+ uptake by root. Our results may enhance understanding of the strategy of applying K+ fertilizer to mitigate crop NH4+ toxicity in agriculture.

Identification, Characterization, and Treatment for a Taurine Transporter (SLC6A6) Variant Resulting in Taurine Deficiency and Pathologies in a Consanguineous Family.

More than 200 Pakistani consanguineous families with multiple individuals having recessive visual impairments were evaluated by exome sequencing and genotyping. One of the families, F315, from the Kohat region of Pakistan contained 2 affected individuals with a homozygous deleterious variant Gly399Val (NM_003043.5:c.1196G>T) in the taurine transporter SLC6A6 (MIM:186854) which segregated with the phenotype of progressive retinal degeneration. Gly399 is highly conserved in vertebrates and molecular modeling suggests that the increased bulk of the valine substitution displaces Tyr138 which participates in substrate coordination and transport and is consistent with the hypomorphic variant exhibiting only ~15% of the transport capacity of the native protein as determined by taurine uptake analyses in HEK‐293 cells transiently or stably expressing SLC6A6 or the Gly399Val variant. The reduced function was attributed to a catalytic effect rather than plasma membrane levels and analysis of patient‐derived fibroblasts of affected individuals mirrored these findings. Affected individuals exhibited cone‐rod retinopathy and cardiomyopathy and remarkably taurine supplementation for 24 months resulted in resolution of the cardiomyopathy and clinical stabilization of the retinopathy.Support or Funding InformationProVisu foundation and ERC grant 219968 to S.E.A., and NIH grant DA027845 to L.K.H.

Evaluation of low concentration glyphosate effect on horseradish peroxidase enzyme activity

Among several pesticides used in agriculture, glyphosate herbicide is one of the most employed in crop fields in spite of its recognized toxic effects on human health and on the environment. Consecutive use of glyphosate in crops can cause significantly increase the severity of various diseases, reduced growth, impaired defenses and alter the physiology of plants affecting the susceptibility or tolerance to diseases. Peroxidase enzymes play an important role in some physiological and pathological processes, involving active oxygen species occurring at the level of plasma membrane and cell wall. The aim of this work was to evaluate the effect of glyphosate on the enzymatic activity of horseradish peroxidase. For this purpose, tests were conducted to determine the optimal pH range and then reaction kinetic where was added, the substrate (catechol), mediator (H2O2), glyphosate and enzyme (horseradish peroxidase), varying the substrate concentration glyphosate to assess the activity of this reaction and the influence of herbicide on the enzyme activity. Results demonstrate that glyphosate increases the activity of horseradish peroxidase in low concentration and can decrease the resistance of the plant to the entry of pathogens.

TGF-β Signaling

Transforming growth factor-β (TGF-β) represents an evolutionarily conserved family of secreted polypeptide factors that regulate many aspects of physiological embryogenesis and adult tissue homeostasis. The TGF-β family members are also involved in pathophysiological mechanisms that underlie many diseases. Although the family comprises many factors, which exhibit cell type-specific and developmental stage-dependent biological actions, they all signal via conserved signaling pathways. The signaling mechanisms of the TGF-β family are controlled at the extracellular level, where ligand secretion, deposition to the extracellular matrix and activation prior to signaling play important roles. At the plasma membrane level, TGF-βs associate with receptor kinases that mediate phosphorylation-dependent signaling to downstream mediators, mainly the SMAD proteins, and mediate oligomerization-dependent signaling to ubiquitin ligases and intracellular protein kinases. The interplay between SMADs and other signaling proteins mediate regulatory signals that control expression of target genes, RNA processing at multiple levels, mRNA translation and nuclear or cytoplasmic protein regulation. This article emphasizes signaling mechanisms and the importance of biochemical control in executing biological functions by the prototype member of the family, TGF-β.