Subcellular and organellar mechanisms have manifested a prominent importance for a broad variety of processes that maintain cellular life at its most basic level. Mammalian two-pore channels (TPCs) ...appear to be cornerstones of these processes in endo-lysosomes by controlling delicate ion-concentrations in their interiors. With evolutionary remarkable architecture and one-of-a-kind selectivity filter, TPCs are an extremely attractive topic
per se.
In the light of the current COVID-19 pandemic, hTPC2 emerges to be more than attractive. As a key regulator of the endocytosis pathway, it is potentially essential for diverse viral infections in humans, as demonstrated. Here, by means of multiscale molecular simulations, we propose a model of sodium transport from the lumen to the cytosol where the central cavity works as a reservoir. Since the inhibition of hTPC2 is proven to stop SARS-CoV2
in vitro
, shedding light on the hTPC2 function and mechanism is the first step towards the selection of potential inhibiting candidates.
The central cavity of hTPC2 works as a reservoir of sodium ions and its occupation modulates the ionic current.
Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe
complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the ...inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.
In the last decade two-pore intracellular channels (TPCs) attracted the interest of researchers, still some key questions remain open. Their importance for vacuolar (plants) and endo-lysosomal ...(animals) function highlights them as a very attractive system to study, both theoretically and experimentally. Indicated as key players in the trafficking of the cell, today they are considered a new potential target for avoiding virus infections, including those from coronaviruses. A particular boost for theoretical examinations has been made with recent high-resolution X-ray and cryo-EM structures. These findings have opened the way for efficient and precise computational studies at the atomistic level. Here we report a set of multiscale-calculations performed on the mTPC1, a ligand- and voltage-gated sodium selective channel. The molecular dynamics and enhanced molecular dynamics simulations were used for a thorough analysis of the mammalian TPC1 behaviour in the presence and absence of the ligand molecule, with a special accent on the supposed bottleneck, the hydrophobic gate. Moreover, from the reconstructed free energy obtained from enhanced simulations, we have calculated the macroscopic conductance of sodium ions through the mTPC1, which we compared with measured single-channel conductance values. The hydrophobic gate works as a steric barrier and the key parameters are its flexibility and the dimension of the sodium first hydration shell.
The flexibility of the pore and the size of sodium hydration shell are key parameters in modulating transport in TPC1.
Passive transport of molecules through nanopores is characterized by the interaction of molecules with pore internal walls and by a general crowding effect due to the constricted size of the nanopore ...itself, which limits the presence of molecules in its interior. The molecule–pore interaction is treated within the diffusion approximation by introducing the potential of mean force and the local diffusion coefficient for a correct statistical description. The crowding effect can be handled within the Markov state model approximation. By combining the two methods, one can deal with complex free energy surfaces taking into account crowding effects. We recapitulate the equations bridging the two models to calculate passive currents assuming a limited occupancy of the nanopore in a wide range of molecular concentrations. Several simple models are analyzed to clarify the consequences of the model. Eventually, a biologically relevant case of transport of an antibiotic molecule through a bacterial porin is used to draw conclusions (i) on the effects of crowding on transport of small molecules through biological channels, and (ii) to demonstrate its importance for modelling of cellular transport.
Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (K
3.1) is highly expressed in the plasma membrane and in the inner ...mitochondrial membrane (mitoK
3.1) of various cancer cell lines. The role mitoK
3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity K
3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC
in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane K
3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoK
3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoK
3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial K
3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.
Human endo-lysosomes possess a class of proteins called TPC channels on their membrane, which are essential for proper cell functioning. This protein family can be functionally studied by expressing ...them in plant vacuoles. Inhibition of hTPC activity by naringenin, one of the main flavonoids present in the human diet, has the potential to be beneficial in severe human diseases such as solid tumor development, melanoma, and viral infections. We attempted to identify the molecular basis of the interaction between hTPC2 and naringenin, using ensemble docking on molecular dynamics (MD) trajectories, but the specific binding site remains elusive, posing a challenge that could potentially be addressed in the future by increased computational power in MD and the combined use of microscopy techniques such as cryo-EM.
COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the world. The viral ...positive-sense single-stranded RNA encodes 31 proteins among which the spike (S) is undoubtedly the best known. Recently, protein E has been reputed as a potential pharmacological target as well. It is essential for the assembly and release of the virions in the cell. Literature describes protein E as a voltage-dependent channel with preference towards monovalent cations whose intracellular expression, though, alters Ca
homeostasis and promotes the activation of the proinflammatory cascades. Due to the extremely high sequence identity of SARS-CoV-2 protein E (E-2) with the previously characterized E-1 (i.e., protein E from SARS-CoV) many data obtained for E-1 were simply adapted to the other. Recent solid state NMR structure revealed that the transmembrane domain (TMD) of E-2 self-assembles into a homo-pentamer, albeit the oligomeric status has not been validated with the full-length protein. Prompted by the lack of a common agreement on the proper structural and functional features of E-2, we investigated the specific mechanism/s of pore-gating and the detailed molecular structure of the most cryptic protein of SARS-CoV-2 by means of MD simulations of the E-2 structure and by expressing, refolding and analyzing the electrophysiological activity of the transmembrane moiety of the protein E-2, in its full length. Our results show a clear agreement between experimental and predictive studies and foresee a mechanism of activity based on Ca
affinity.
A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various ...human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.
We investigated, by using all‐atom molecular dynamics simulations, the effect of the outer membrane of Gram‐negative bacteria, composed in the outer leaflet by polar/charged lipopolysaccharides ...(LPS), on the electrostatic properties of general porins from the Enterobacteriaceae family. General porins constitute the main path for the facilitated diffusion of polar antibiotics through the outer membrane. As model system we selected OmpK36 from Klebsiella pneumoniae, the ortholog of OmpC from Escherichia coli. This species presents high variability of amino acid composition of porins, with the effect to increase its resistance to the penetration of antibiotics. The various properties we analyzed seem to indicate that LPS acts as an independent layer without affecting the internal electrostatic properties of OmpK36. The only apparent effect on the microsecond time scale we sampled is the appearance of calcium ions, when present at moderate concentration in solution, inside the pore. However, we noticed increased fluctuations of the polarization density and only minor changes on its average value.
We analyzed structural and electrostatic properties of the porin OmpK36 within various membrane environments, including the lipopolysaccharide from Klebsiella pneumoniae and Escherichia coli, and a POPC bilayer. Our results reveal distinct influences of the different environments: while the average value of the transversal electric field remains relatively constant, the environments modulate its fluctuations, suggesting a subtle role of the surrounding.
The extremophile bacterium D. radiodurans boasts a distinctive cell envelope characterized by the regular arrangement of three protein complexes. Among these, the Type II Secretion System (T2SS) ...stands out as a pivotal structural component. We used cryo-electron microscopy to reveal unique features, such as an unconventional protein belt (DR_1364) around the main secretin (GspD), and a cap (DR_0940) found to be a separated subunit rather than integrated with GspD. Furthermore, a novel region at the N-terminus of the GspD constitutes an additional second gate, supplementing the one typically found in the outer membrane region. This T2SS was found to contribute to envelope integrity, while also playing a role in nucleic acid and nutrient trafficking. Studies on intact cell envelopes show a consistent T2SS structure repetition, highlighting its significance within the cellular framework.
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