June 2024
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18 Reads
Journal of Molecular Biology
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June 2024
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18 Reads
Journal of Molecular Biology
May 2024
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62 Reads
Nature Communications
Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K⁺ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K⁺ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
December 2023
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156 Reads
Ceramides are bioactive sphingolipids that play pivotal roles in regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by a family of six ceramide synthase enzymes (CerS), each with distinct specificity for the acyl-CoA substrate. Importantly, the acyl chain length plays a key role in determining the physiological function of ceramides, as well as their role in metabolic disease. Ceramide with an acyl chain length of 16 carbons (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease, and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here, we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, together with biochemical characterization using intact protein and small molecule mass spectrometry, reveal that CerS catalysis proceeds via a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B 1 , providing new insights into its inhibition of CerS. These results provide a framework for understanding the mechanisms of CerS function, selectivity, and inhibition, and open new directions for future drug discovery targeting the ceramide and sphingolipid pathways.
October 2023
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77 Reads
Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, we generated camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identified selective binders including several that directly modulate channel activity. Crystal structures of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These tools therefore provide important insights into TREK channel gating and a more selective approach to the modulation of K2P channel activity via their extracellular domains.
May 2023
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119 Reads
Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes which link substrate release and opening of the cytoplasmic gate, and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
October 2022
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328 Reads
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11 Citations
Nature Genetics
Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the ‘X-gate’, a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies. Heterozygous de novo gain-of-function mutations in KCNK3, which encodes the two-pore-domain K+ channel TASK-1, cause a channelopathy characterized by developmental delay with sleep apnea.
August 2021
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181 Reads
Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its reported link with sleep apnea remains poorly understood. Here we describe a novel developmental disorder with sleep apnea caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the X-gate, a gating motif which controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein coupled receptor pathways but which can be inhibited by several clinically relevant drugs. These findings demonstrate a clear role for TASK-1 in sleep apnea and identify possible therapeutic strategies.
June 2021
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351 Reads
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56 Citations
Nature Structural & Molecular Biology
Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson’s, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy. ELOVLs are membrane-embedded enzymes that elongate very long chain fatty acids, precursors of sphingolipids and ceramides. The first crystal structure of a human ELOVL reveals an unexpected reaction mechanism, suggesting potential approaches for inhibition in disease.
May 2021
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218 Reads
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19 Citations
The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.
May 2021
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151 Reads
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33 Citations
Science Translational Medicine
Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.
... A predicted human ortholog of TWK-26 is KCNK3/TASK, a two-pore domain K + leak channel that is broadly expressed, including in the brain and small intestine, and that is strongly inhibited by extracellular acidification (Duprat et al. 1997). Interestingly, KCNK3 gain-of-function mutations increase channel opening probability and are associated with developmental disorders with sleep apnea (Sörmann et al. 2022). KCNK3 contains a structural motif called the X-gate, which regulates the opening and closing of the channel (Rödström et al. 2020). ...
October 2022
Nature Genetics
... The Elovl sequence retrieval strategy used in the present study revealed that P. dumerilii has six distinct elovl genes (EloA-F). The analysis of the deduced amino acid protein sequences of the so-called 'EloE' and 'EloF' showed that these Elovl do not satisfy with all the characteristics shared among PUFA elongases [51,74]. Consistently, the phylogenetic analysis clearly established that the P. dumerilii EloE and EloF clustered with Elovl3 and Elovl6, enzymes with roles in the elongation of non-PUFA substrates [71]. ...
June 2021
Nature Structural & Molecular Biology
... To investigate the mechanistic basis for phosphorylation regulation of TREK channels we first tested if the (de-)phosphorylated states with enhanced or diminished activity can be assigned to either the upor down-state conformation of TREK utilizing as before the statesensitive inhibitor NFx ( Fig. 4a) 14,20,47 . We measured TREK-1 channel currents in response to 800 ms voltage ramps (− 80 mV to + 80 mV) in excised membrane patches under asymmetrical K + conditions (Fig. 4b). ...
May 2021
... The parameters were calculated using the Seahorse Wave Desktop software from Agilent. Respiratory measurements of cytosolic mitochondria isolated from mouse liver were performed as described previously (82). Briefly, 5 μg of mitochondrial suspension was seeded onto a Seahorse XFe96 plate (Agilent, 103792-100) to measure the pyruvate+malatedriven respiration (complex I) and 4 μg for the palmitoyl-carnitinedriven respiration (β-oxidation). ...
May 2021
Science Translational Medicine
... The maturation of lamin A involves post-translational modifications performed on the prelamin A precursor, leading to the final essential cleavage step mediated by . ZMPSTE24 recognizes farnesylated prelamin A by its zinc metalloprotease motif and catalyses an endoproteolytic cleavage of its C-terminal residues to form mature lamin A [10][11][12] . Thus, farnesylated prelamin A under normal conditions is a transient intermediate form that is essentially undetectable in cells because of its efficient conversion to lamin A. ...
December 2020
Journal of Biological Chemistry
... Several recent reports have shown that C. elegans and mammalian cells need it to accumulate fat, and that disrupting the gene for TMEM120A leads to metabolic defects in mice fed a high-fat diet (Czapiewski et al., 2021;Li et al., 2021). In addition, its structure also resembles that of ELOVL7, a membrane-embedded enzyme that helps to elongate fatty acids (Nie et al., 2020;Niu et al., 2021;Xue et al., 2021). However, TMEM120A does not catalyze the same reactions as ELOVL7, and the possible substrates and end products of TMEM120A remain unknown (Niu et al., 2021). ...
Reference:
Pain or gain?
November 2020
... To try and elucidate further how treprostinil might be conferring its inhibitory effects on the TREK channels, we considered a known gain-of-function (GOF) mutation that affects the gating of an alanine in TREK-1, resulted in channels with large outward currents of 95.8 pA pF −1 (95% CI: 74.6 to 117.0, n 5) which were significantly larger (p < 0.05, unpaired t-test) than WT TREK-1, 24.2 pA pF −1 (95% CI: 18.3 to 30.1, n 8) and in agreement with channels having a higher P o (Proks et al., 2020). Application of 1 µM treprostinil was found to still significantly reduce (p < 0.05, FIGURE 4 | Effect of treprostinil on mutated TREK-2/L320A, TREK-1/L289A and TREK-2/K302Q (A) Measurement of whole-cell TREK-2/L320A current (pA pF −1 ) in control (n 6) and following application of treprostinil (1 μM, n 6, **p 0.003; paired t-test) (B) Comparison of the inhibition of WT TREK-2 and TREK-2/L320A current by treprostinil (1 µM) calculated as the difference of current measured in control, with that measured after exposure to treprostinil, expressed as a percentage, displayed as a Box and Whiskers plot. ...
October 2020
... According to the AntiSMASH analysis of S. vitiensis genome sequence (GenBank NZ_KB900388), no secondary metabolite BGCs are detected in this region ( Proteins of this type are typically one of the components of transport complexes of the ABC-2 type, which facilitate ATP-mediated transport of one or more diverse substrates. Wellknown examples of such proteins include CcmB, responsible for transporting haeme in Escherichia coli and Mycobacterium tuberculosis, or DrrB in the doxorubicin producer S. peucetius [3]. ...
September 2020
... Defining the properties needed for substrate recognition, portal entry, and positioning of substrate will help clarify our understanding of the mechanism of prelamin A cleavage by ZMPSTE24. We have recently shown through comprehensive mutagenesis of residues surrounding the cleavage site of prelamin A (TRSY^LLGN) that having two hydrophobic residues just C-terminal to the scissile bond in the P1' and P2' positions (the two leucines in wild-type prelamin A) is critical for its cleavage by ZMPSTE24 [24]. In some proteases a region distant from the active site, termed an exosite, can facilitate the capture and proper orientation of substrate for cleavage. ...
August 2020
... In the first step, protein farnesyltransferase catalyzes the addition of a farnesyl moiety to the cysteine [10,11]. Next, the RCE1 protease catalyzes cleavage between the farnesylated cysteine and -AAX residues, which are -SIM in prelamin A (it should be noted that ZMPSTE24 can also cleave some CAAX motifs, but not that of prelamin A) [12]. Third, the carboxyl-terminal farnesylcysteine is methylated in a reaction catalyzed by isoprenylcysteine carboxyl methyltransferase [13,14]. ...
May 2020