Cytosolic AND pH

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt ). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9...

BACKGROUND: Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK ) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins...

Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes' cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations...

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in brain, spinal cord and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe2+ ) generates reactive oxygen species (ROS)...

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways...

Polymeric micelles have been extensively studied as vectors for the delivery of hydrophobic drugs for the treatment of cancers and other diseases. Despite intensive research, few formulations provide significant benefits, and even fewer have been clinically approved. While many traditional non-responsive micelles have excellent safety profiles, they lack the ability to respond to the intracellular environment and release their cargo in a spatiotemporally defined manner to effectively deliver large doses of cytotoxic drugs into the cytosol of cells that overwhelm efflux pumps...

Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials...

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes...

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen...

Small interfering RNA (siRNA) has a promising future in the treatment of ocular diseases due to its high efficiency, specificity, and low toxicity in inhibiting the expression of target genes and proteins. However, due to the unique anatomical structure of the eye and various barriers, delivering nucleic acids to the retina remains a significant challenge. In this study, we rationally design PACD, an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block (A), a siRNA binding block (B) and a pH-responsive block (C)...

Although suppressed cAMP levels have been linked to cancer for nearly five decades, the molecular basis remains uncertain. Here, we identify endosomal pH as a novel regulator of cytosolic cAMP homeostasis and a promoter of transformed phenotypic traits in colorectal cancer (CRC). Combining experiments and computational analysis, we show that the Na+/H+ exchanger NHE9 contributes to proton leak and causes luminal alkalinization, which induces resting [Ca2+], and in consequence, represses cAMP levels, creating a feedback loop that echoes nutrient deprivation or hypoxia...

BACKGROUND: Pulmonary hypertension (PH) is a complex multifactorial vascular pathology characterized by an increased pulmonary arterial pressure, vasoconstriction, remodelling of the pulmonary vasculature, thrombosis in situ and inflammation associated with right-side heart failure. Herein, we explored the potential beneficial effects of treatment with siRNA AP-1 on pulmonary arterial hypertension (PAH), right ventricular dysfunction along with perivascular and interstitial fibrosis in pulmonary artery-PA, right ventricle-RV and lung in an experimental animal model of monocrotaline (MCT)-induced PAH...

Plants rely on precise regulation of their stomatal pores to effectively carry out photosynthesis while managing water status. The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical light signaling repressor, is known to repress stomatal opening, but the exact cellular mechanisms remain unknown. Here, we show that COP1 regulates stomatal movement by controlling the pH levels in guard cells. cop1-4 mutants have larger stomatal apertures and disrupted pH dynamics within guard cells, characterized by increased vacuolar and cytosolic pH and reduced apoplastic pH, leading to abnormal stomatal responses...

The potential role of astrocytes in lateral habenula (LHb) in modulating anxiety was explored in this study. The habenula are a pair of small nuclei located above the thalamus, known for their involvement in punishment avoidance and anxiety. Herein, we observed an increase in theta-band oscillations of local field potentials (LFPs) in the LHb when mice were exposed to anxiety-inducing environments. Electrical stimulation of LHb at theta-band frequency promoted anxiety-like behavior. Calcium (Ca2+ ) levels and pH in the cytosol of astrocytes and local brain blood volume changes were studied in mice expressing either a Ca2+ or a pH sensor protein specifically in astrocytes and mScarlet fluorescent protein in the blood plasma using fiber photometry...

Potassium (K+ ) is the most abundant cation in the cytosol and is maintained at high concentrations within the mitochondrial matrix through potassium channels. However, many effects of K+ at such high concentrations on mitochondria and the underlying mechanisms remain unclear. This study aims to elucidate these effects and mechanisms by employing fluorescence imaging techniques to distinguish and precisely measure signals inside and outside the mitochondria. We stained the mitochondrial matrix with fluorescent dyes sensitive to K+ , pH, reactive oxygen species (ROS), and membrane potential in plasma membrane-permeabilized C6 cells and isolated mitochondria from C6 cells...

Salinity is one of the most dangerous types of stress in agriculture. Acting on the root, salinity causes changes in physiological processes in the shoot, especially photosynthesis, which is crucial for plant productivity. In our study, we used potato plants, the most important crop, to investigate the role of salt-induced signals in changes in photosynthesis activity. We found a salt-induced polyphasic decrease in photosynthesis activity, and the earliest phase started several minutes after salt addition. We found that salt addition triggered rapid hydraulic and calcium waves from root to shoot, which occurred earlier than the first phase of the photosynthesis response...

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability. Escherichia coli strains Origami B and BL21 (DE3) were selected for their distinct cytosolic environment to express BhChitA chitinase of Bacillus halodurans C-125 and to investigate the role of disulfide bond formation and proper folding on its stability and activity...

BACKGROUND: Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+ -activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1β release...

BACKGROUND: Drosophila serve as exceptional alternative models for in vivo and ex vivo research and may provide an avenue for in-depth investigation for human ClC-5 and Dent disease type 1 (DD1). The Drosophila ClC-c (CG5284) has sequence homology with human ClC-5 and is hypothesized to encompass similar functional and phenotypical roles with ClC-5 and variants that cause DD1. METHODS: Ion transport function and activity of Drosophila ClC-c and homologous DD1 variants were assessed by voltage clamp electrophysiology...

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH...