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The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H + ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl - /H + antiporter responsible for lysosomal Cl - permeability, is a candidate to contribute to the acidification process as part of this ‘counterion pathway’ The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here, we demonstrate that depleting PI(3,5)P2 by inhibiting the kinase PIKfyve causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together, these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation. The TRPV1 ion channel is a heat-sensing receptor that is also activated by vanilloid compounds, but the molecular underpinnings of thermosensing have remained elusive. Here authors use in solution NMR on the isolated human TRPV1 S1-S4 domain and show that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis 1 – 3 . Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate 4 , 5 . Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5 ) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor—which binds to the same site as citrate—arrests the transport cycle of NaCT. The NaCT–inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy) 6 – 8 . Structures of the human sodium-dependent citrate transporter NaCT in complexes with citrate or a small-molecule inhibitor reveal how the inhibitor—which binds to the same site as citrate—arrests the transport cycle of NaCT.

Warfarin has been the most widely prescribed anticoagulant for decades. It functions by inhibiting the membrane enzyme vitamin K epoxide reductase (VKOR), but the molecular details of this effect have remained elusive. Two new studies shed light on the warfarin-VKOR interaction. The work has implications for precision medicine and could guide drug discovery.

Lysosomes process cellular waste and coordinate responses to metabolic challenge. Central to lysosomal homeostasis are phosphoinositide lipids, key signaling molecules which establish organelle identity, regulate membrane dynamics and are tightly linked to the pathophysiology and therapy of lysosomal storage disorders, neurodegeneration, and cancer. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) interacts with multiple lysosomal membrane proteins and plays a critical role in regulating lysosomal pH by directly inhibiting the chloride/proton antiporter ClC-7, though the molecular mechanism of this inhibition remains unclear. Here, using a combination of functional, structural, and computational analysis, we demonstrate that PI(3,5)P2 binding dramatically remodels the structure of ClC-7 by inducing close association between cytosolic and transmembrane domains. Disease-causing mutations show increased transport activity through loss of PI(3,5)P2 binding and subsequent inhibition. Conversely, ClC-7 activation is correlated with dissociation and increased disorder of the cytoplasmic domain along with novel transmembrane domain conformations, revealing a mechanistic link between specific lysosomal lipids, transporter regulation, and the enigmatic basis of the ClC-7 slow gate.

Protein dynamics have emerged as a key feature associated with function in various systems. Here, NMR-based studies coupled with computational cheminformatics and cellular function are leveraged to identify a relationship between the human cold and menthol receptor TRPM8 dynamics, chemical structure, and cellular potency. TRPM8 is a validated target for a variety of pain indications but generally has been clinically limited by on-target side effects impacting thermosensing and thermoregulation. This study shows that cheminformatic analysis of a TRPM8 regulating small molecule ligand library correlates with cellular function. Electrophysiology studies further validate the relationship and show a correlation between chemical structure and functional features such as compound potency. Solution NMR studies of the TRPM8 voltage sensing-like domain, which houses the canonical menthol ligand binding site, show that ligand binding conformationally selects NMR-detected TRPM8 dynamics in a manner that quantitatively correlates with chemical structure. The relationship between chemical structure and protein dynamics can be used predictively, where a chemical structure is predictive of dynamics in a latent reduced dimensionality space. Moreover, the robustness of the conformational selection of the dynamic ensemble is evaluated by varying related and divergent chemotypes, signal-to-noise sensitivity, and sample bias. Taken together, this study identifies that protein dynamics can serve as a quantifiable bridge between chemical structure and cellular function, which has implications for drug discovery in difficult systems.

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H.sup.+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl.sup.-/H.sup.+ antiporter responsible for lysosomal Cl.sup.- permeability, is a candidate to contribute to the acidification process as part of this 'counterion pathway' The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here, we demonstrate that depleting PI(3,5)P2 by inhibiting the kinase PIKfyve causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together, these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. However, the role of the S1-S4 domain in thermosensing is unclear. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, Far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this “counterion pathway”. The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation. Competing Interest Statement The authors have declared no competing interest.

Prospect bowler Amy Ward likely bowled the state's highest six- game series Saturday in the Prospect Invitational, but you won't find it in the Illinois High School Association record books. The state doesn't keep six-game series records, but Prospect coach Greg Troyer says he is confident Ward's score of 1,408 is the highest in state history. Lakewood (Ohio) St. Edward Quintangular: Providence took it's No. 1 national wrestling ranking to the Cleveland suburb of Lakewood and got a lesson in a 34-16 defeat. Providence was No. 1 in several national polls, while St. Edward was ranked fourth in the same polls.