Valeria Jaramillo-Martinez

Electrostatic interactions often play a role in determining the thermodynamic and kinetic properties of protein-protein interactions. However, the role of long-range electrostatic interactions in intrinsically disordered protein (IDP) binding is less clear, as they often bind in multiple steps including initial formation of a disordered encounter complex, followed by rearrangement into the bound state. We varied the salt concentration to probe the role of long-range electrostatic interactions in the binding of the highly charged AbpSH3 domain and the oppositely charged IDP ArkA. Using isothermal titration calorimetry, we observe that salt enthalpically destabilizes the bound complex. Molecular dynamics and NMR experiments reveal that salt has little effect on the bound state structure. However, simulations show that salt destabilizes the encounter complex intermediate, which primarily affects the association rate as measured by NMR. Consistent with these results, salt has the largest stabilizing effect on the apo SH3 domain, as cations substitute for the transient and long-range electrostatic interactions that can form with ArkA in the complex. We reveal a detailed picture of how a highly charged domain uses long-range, fuzzy, electrostatic interactions to help reach the bound state, a mechanism that is likely common among other highly charged domains that bind IDPs. TOC Image:

The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na ⁺ gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. We mechanistically classify the molecular phenotypes of six mutations. C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. G219R, S427L, and L488P are hampered by low protein expression, ER retention, and reduced transport. Mutants’ mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. These experiments provide a comprehensive understanding of the mutation’s defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different treatment approaches to either restore transport function, or enable correction of protein folding defects. Summary: Loss-of-function mutations in the SLC13A5 causes SLC13A5-Epilepsy, a devastating disease characterized by neonatal epilepsy. Currently no cure is available. We clarify the molecular-level defects to guide future developments for phenotype-specific treatment of disease-causing mutations.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases the cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced the expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here, we found SLC7A11 to be inhibited by niclosamide with an IC₅₀ value in the range of 0.1–0.2 μM. In addition to the direct inhibition of SLC38A5 and SLC7A11, the pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased the glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here we found SLC7A11 to be inhibited by niclosamide with an IC50 value in the range of 0.1-0.2 M. In addition to the direct inhibition of SLC38A5 and SLC7A11, pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

Sigma receptors are non-opiate/non-phencyclidine receptors that bind progesterone and/or heme and also several unrelated xenobiotics/chemicals. They reside in the plasma membrane and in the membranes of the endoplasmic reticulum, mitochondria, and nucleus. Until recently, the biology/pharmacology of these proteins focused primarily on their role in neuronal functions in the brain/retina. However, there have been recent developments in the field with the discovery of unexpected roles for these proteins in iron/heme homeostasis. Sigma receptor 1 (S1R) regulates the oxidative stress-related transcription factor NRF2 and protects against ferroptosis, an iron-induced cell death process. Sigma receptor 2 (S2R), which is structurally unrelated to S1R, complexes with progesterone receptor membrane components PGRMC1 and PGRMC2. S2R, PGRMC1, and PGRMC2, either independently or as protein–protein complexes, elicit a multitude of effects with a profound influence on iron/heme homeostasis. This includes the regulation of the secretion of the iron-regulatory hormone hepcidin, the modulation of the activity of mitochondrial ferrochelatase, which catalyzes iron incorporation into protoporphyrin IX to form heme, chaperoning heme to specific hemoproteins thereby influencing their biological activity and stability, and protection against ferroptosis. Consequently, S1R, S2R, PGRMC1, and PGRMC2 potentiate disease progression in hemochromatosis and cancer. These new discoveries usher this intriguing group of non-traditional progesterone receptors into an unchartered territory in biology and medicine.

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye–Huckel screening and a nonspecific territorial ion‐binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short‐range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years.

Niclosamide, a drug used to treat tapeworm infection, possesses anticancer effects by interfering with multiple signaling pathways. Niclosamide also causes intracellular acidification. We have recently discovered that the amino acid transporter SLC38A5, an amino acid-dependent Na⁺/H⁺ exchanger, activates macropinocytosis in cancer cells via amino acid-induced intracellular alkalinization. Therefore, we asked whether niclosamide will block basal and SLC38A5-mediated macropinocytosis via intracellular acidification. We monitored macropinocytosis in pancreatic and breast cancer cells using TMR-dextran and the function of SLC38A5 by measuring Li⁺-stimulated serine uptake. The peptide transporter activity was measured by the uptake of glycylsarcosine. Treatment of the cancer cells with niclosamide caused intracellular acidification. The drug blocked basal and serine-induced macropinocytosis with differential potency, with an EC₅₀ of ~5 μM for the former and ~0.4 μM for the latter. The increased potency for amino acid-mediated macropinocytosis is due to direct inhibition of SLC38A5 by niclosamide in addition to the ability of the drug to cause intracellular acidification. The drug also inhibited the activity of the H⁺-coupled peptide transporter. We conclude that niclosamide induces nutrient starvation in cancer cells by blocking macropinocytosis, SLC38A5 and the peptide transporter. These studies uncover novel, hitherto unknown, mechanisms for the anticancer efficacy of this antihelminthic.

INDY (I’m Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate²⁻ in exchange for other dicarboxylates, and the latter being a Na⁺-coupled uniporter for citrate²⁻. Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues