Fusion of human monocarboxylate transporter 1 with basigin and expression in S. cerevisiae: Is basigin more than a chaperone?

Lactate is a key metabolite in human cells. A regulated transport across membranes is vital for cellular function while deregulated transport is a hallmark of cancer. In tumor cells, glycolysis as the main route for ATP synthesis even in the presence of oxygen (Warburg effect) demands rapid lactate clearance to avoid acidification. Oxygenic cancer cells, in turn, rely on an efficient retrieval of extracellular lactate to fuel the citric acid cycle (reverse Warburg effect). To maintain an active interchange between cells, four monocarboxylate transporters (MCT1−4) manage bi-directional, proton-coupled transport across plasma membranes. For its translocation to the plasma membrane, MCT1 demands chaperoning by a member of the immunoglobulin superfamily, namely basigin. Both proteins remain complexed at the plasma membrane. Although frequently suggested, a direct effect of the chaperone on MCT1-mediated transport is not resolvable in commonly used expression systems. In this study, MCT1 expression in S. cerevisiae Δjen1 Δady2 profited from a basigin-independent translocation in a system with zero background from endogenous monocarboxylate transporters or basigin homologs. The molecular fusion with truncated basigin constructs revealed an effect on transmembrane L-lactate distribution at the domain level. In zero-trans influx experiments using 14C-labeled substrate, the presence of basigin’s extracellular Ig-I domain permitted a 4.5-fold intracellular L-lactate accumulation in the transport equilibrium. At near-neutral pH, cytosolic L-lactate concentrations greatly exceeded those provided with the buffer. The absence of the basigin Ig-I domain due to truncation or misfolding reversed this effect. The identification of patches of positive and negative surface potentials and evidence from charge-resolving point mutations indicated an electrostatic attraction of L-lactate anions and protons. This thesis deduces a substrate harvesting function of basigin that creates a “microenvironment” of locally increased concentrations and drives L-lactate influx according to Le Chatelier’s principle. This influx was physiologically relevant and promoted cell growth on L-lactate medium. According to classical and reverse Warburg effects, highly adapted tumor cells require a fine-tuned transmembrane L-lactate distribution and basigin might be an important determinant. Hereof, MCTs are promising targets in the anti-tumor therapy. The basigin-MCT1 fusion set-up from this study further revealed two known MCT1 inhibitors, AZD3965 and p-chloromercuribenzene sulfonate (pCMBS), as direct and basigin-independent modifiers. Cys159 in the transporter cavity was revealed as selectively targetable by pCMBS leading to a complete transport inhibition. Smaller cysteine-modifiers had a less prominent effect and lacked site-specificity. Cys159 is proposed to constitute a hinge region of the alternating access transporter and a wedge-like modification locks MCT1 in the outward open conformation. This reveals a target region for inhibitor design and in the future, Cys159 might serve as a natural anchor to introduce distinct labels and report on physicochemical modalities in a most critical part of the transporter

Lipid-Iron Nanoparticle with a Cell Stress Release Mechanism Combined with a Local Alternating Magnetic Field Enables Site-Activated Drug Release

Simple Summary A novel active release system magnetic sphingomyelin-containing liposome encapsulated with indocyanine green, fluorescent marker, or the anticancer drug cisplatin was evaluated. The liposomal sphingomyelin is a target for the sphingomyelinase enzyme, which is released by stressed cells. Thus, sphingomyelin containing liposomes behave as a sensitizer for biological stress situations. In addition, the liposomes were engineered by adding paramagnetic beads to act as a receiver of outside given magnetic energy. The enzymatic activity towards liposomes and destruction caused by the applied magnetic field caused the release of the content from the liposomes. By using these novel liposomes, we could improve the drug release feature of liposomes. The improved targeting and drug-release were shown in vitro and the orthotopic tongue cancer model in mice optical imaging. The increased delivery of cisplatin prolonged the survival of the targeted delivery group versus free cisplatin. Most available cancer chemotherapies are based on systemically administered small organic molecules, and only a tiny fraction of the drug reaches the disease site. The approach causes significant side effects and limits the outcome of the therapy. Targeted drug delivery provides an alternative to improve the situation. However, due to the poor release characteristics of the delivery systems, limitations remain. This report presents a new approach to address the challenges using two fundamentally different mechanisms to trigger the release from the liposomal carrier. We use an endogenous disease marker, an enzyme, combined with an externally applied magnetic field, to open the delivery system at the correct time only in the disease site. This site-activated release system is a novel two-switch nanomachine that can be regulated by a cell stress-induced enzyme at the cellular level and be remotely controlled using an applied magnetic field. We tested the concept using sphingomyelin-containing liposomes encapsulated with indocyanine green, fluorescent marker, or the anticancer drug cisplatin. We engineered the liposomes by adding paramagnetic beads to act as a receiver of outside magnetic energy. The developed multifunctional liposomes were characterized in vitro in leakage studies and cell internalization studies. The release system was further studied in vivo in imaging and therapy trials using a squamous cell carcinoma tumor in the mouse as a disease model. In vitro studies showed an increased release of loaded material when stress-related enzyme and magnetic field was applied to the carrier liposomes. The theranostic liposomes were found in tumors, and the improved therapeutic effect was shown in the survival studies.Peer reviewe

Basigin drives intracellular accumulation of l-lactate by harvesting protons and substrate anions.

Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant

Utilizing Sphingomyelinase Sensitizing Liposomes in Imaging Intestinal Inflammation in Dextran Sulfate Sodium-Induced Murine Colitis

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gastrointestinal tract, resulting in severe symptoms. At the moment, the goal of medical treatments is to reduce inflammation. IBD is treated with systemic anti-inflammatory compounds, but they have serious side effects. The treatment that is most efficient and causes the fewest side effects would be the delivery of the drugs on the disease site. This study aimed to investigate the suitability of sphingomyelin (SM) containing liposomes to specifically target areas of inflammation in dextran sulfate sodium-induced murine colitis. Sphingomyelin is a substrate to the sphingomyelinase enzyme, which is only present outside cells in cell stress, like inflammation. When sphingomyelin consisting of liposomes is predisposed to the enzyme, it causes the weakening of the membrane structure. We demonstrated that SM-liposomes are efficiently taken up in intestinal macrophages, indicating their delivery potential. Furthermore, our studies showed that sphingomyelinase activity and release are increased in a dextran sulfate sodium-induced IBD mouse model. The enzyme appearance in IBD disease was also traced in intestine samples of the dextran sulfate sodium-treated mice and human tissue samples. The results from the IBD diseased animals, treated with fluorescently labeled SM-liposomes, demonstrated that the liposomes were taken up preferentially in the inflamed colon. This uptake efficiency correlated with sphingomyelinase activity