Journal articles: 'HTREK1'

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Miller, Paula, Chris Peers, and Paul J. Kemp. "Polymodal regulation of hTREK1 by pH, arachidonic acid, and hypoxia: physiological impact in acidosis and alkalosis." American Journal of Physiology-Cell Physiology 286, no. 2 (February 2004): C272—C282. http://dx.doi.org/10.1152/ajpcell.00334.2003.

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Expression of the human tandem P domain K+ channel, hTREK1, is limited almost exclusively to the central nervous system, where ambient Po2 can be as low as 20 Torr. We have previously shown that this level of hypoxia evokes a maximal inhibitory influence on recombinant hTREK1 and occludes the activation by arachidonic acid; this has cast doubt on the idea that TREK1 activation during brain ischemia could facilitate neuroprotection via hyperpolarizing neurons in which it is expressed. Using both whole cell and cell-attached patch-clamp configurations, we now show that the action of another potent TREK activator and ischemia-related event, intracellular acidification, is similarly without effect during compromised O2 availability. This occlusion is observed in either recording condition, and even the concerted actions of both arachidonic acid and intracellular acidosis are unable to activate hTREK1 during hypoxia. Conversely, intracellular alkalinization is a potent channel inhibitor, and hypoxia does not reverse this inhibition. However, increases in intracellular pH are unable to occlude either arachidonic acid activation or hypoxic inhibition. These data highlight two important points. First, during hypoxia, modulation of hTREK1 cannot be accomplished by parameters known to be perturbed in brain ischemia (increased extracellular fatty acids and intracellular acidification). Second, the mechanism of regulation by intracellular alkalinization is distinct from the overlapping structural requirements known to exist for regulation by arachidonic acid, membrane distortion, and acidosis. Thus it seems likely that hTREK1 regulation in the brain will be physiologically more relevant during alkalosis than during ischemia or acidosis.

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Miller, P., P. J. Kemp, and C. Peers. "Structural requirements for O2 sensing by the human tandem-P domain channel, hTREK1." Biochemical and Biophysical Research Communications 331, no. 4 (June 2005): 1253–56. http://dx.doi.org/10.1016/j.bbrc.2005.04.042.

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Metri, Vishal, Swagata Ghatak, Soumyendu Raha, and S. K. Sikdar. "Patch clamp data driven stochastic modeling and simulation of hTREK1 potassium ion channel gating." Chemical Physics 516 (January 2019): 182–90. http://dx.doi.org/10.1016/j.chemphys.2018.07.030.

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El Hachmane, Mickael-F., Kathryn A. Rees, Emma L. Veale, Vadim V. Sumbayev, and Alistair Mathie. "Enhancement of TWIK-related Acid-sensitive Potassium Channel 3 (TASK3) Two-pore Domain Potassium Channel Activity by Tumor Necrosis Factor α." Journal of Biological Chemistry 289, no. 3 (December 4, 2013): 1388–401. http://dx.doi.org/10.1074/jbc.m113.500033.

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TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2–15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.

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Nayak, Tapan Kumar, Saswati Dana, Soumyendu Raha, and Sujit K. Sikdar. "Activator-induced dynamic disorder and molecular memory in human two-pore domain hTREK1 K+ channel." Journal of Chemical Biology 4, no. 2 (February 1, 2011): 69–84. http://dx.doi.org/10.1007/s12154-010-0053-3.

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Stueber, Thomas, Mirjam J. Eberhardt, Christoph Hadamitzky, Annette Jangra, Stefan Schenk, Felicia Dick, Carsten Stoetzer, et al. "Quaternary Lidocaine Derivative QX-314 Activates and Permeates Human TRPV1 and TRPA1 to Produce Inhibition of Sodium Channels and Cytotoxicity." Anesthesiology 124, no. 5 (May 1, 2016): 1153–65. http://dx.doi.org/10.1097/aln.0000000000001050.

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Abstract Background The relatively membrane-impermeable lidocaine derivative QX-314 has been reported to permeate the ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) to induce a selective inhibition of sensory neurons. This approach is effective in rodents, but it also seems to be associated with neurotoxicity. The authors examined whether the human isoforms of TRPV1 and TRPA1 allow intracellular entry of QX-314 to mediate sodium channel inhibition and cytotoxicity. Methods Human embryonic kidney 293 (HEK-293) cells expressing wild-type or mutant human (h) TRPV1 or TRPA1 constructs as well as the sodium channel Nav1.7 were investigated by means of patch clamp and ratiometric calcium imaging. Cytotoxicity was examined by flow cytometry. Results Activation of hTRPA1 by carvacrol and hTRPV1 by capsaicin produced a QX-314–independent reduction of sodium current amplitudes. However, permeation of QX-314 through hTRPV1 or hTRPA1 was evident by a concentration-dependent, use-dependent inhibition of Nav1.7 activated at 10 Hz. Five and 30 mM QX-314 activated hTRPV1 via mechanisms involving the intracellular vanilloid-binding domain and hTRPA1 via unknown mechanisms independent of intracellular cysteins. Expression of hTRPV1, but not hTRPA1, was associated with a QX-314–induced cytotoxicity (viable cells 48 ± 5% after 30 mM QX-314) that was ameliorated by the TRPV1 antagonist 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (viable cells 81 ± 5%). Conclusions The study data demonstrate that QX-314 directly activates and permeates the human isoforms of TRPV1 and TRPA1 to induce inhibition of sodium channels, but also a TRPV1-dependent cytotoxicity. These results warrant further validation of this approach in more intact preparations and may be valuable for the development of this concept into clinical practice.

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Gil, V., D. Gallego, H. Moha Ou Maati, R. Peyronnet, M. Martínez-Cutillas, C. Heurteaux, M. Borsotto, and M. Jiménez. "Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon." American Journal of Physiology-Gastrointestinal and Liver Physiology 303, no. 3 (August 1, 2012): G412—G423. http://dx.doi.org/10.1152/ajpgi.00040.2012.

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Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and Nω-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

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Wiedmann, Felix, Daniel Schlund, Francisco Faustino, Manuel Kraft, Antonius Ratte, Dierk Thomas, Hugo A. Katus, and Constanze Schmidt. "N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels." International Journal of Molecular Sciences 20, no. 20 (October 20, 2019): 5193. http://dx.doi.org/10.3390/ijms20205193.

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Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

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Oates, David, Wendy Nice, Roy Taylor, and Wendy Hanson. "Porous ceramics for gas turbine applications." High Temperatures-High Pressures 27/28, no. 3 (1995): 339–45. http://dx.doi.org/10.1068/htrt01.

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Dozhdikov, Vitaly, and Vadim Petrov. "New automated apparatus for the measurement of spectral emissivity of nonconducting materials by high-speed spectrometer." High Temperatures-High Pressures 27/28, no. 4 (1995): 403–10. http://dx.doi.org/10.1068/htrt41.

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Giaretto, Valter, Elio Miraldi, and Giuseppe Ruscica. "Simultaneous estimations of radiative and conductive properties in lightweight insulating materials." High Temperatures-High Pressures 27/28, no. 2 (1995): 191–204. http://dx.doi.org/10.1068/htrt51.

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Gryksa, Werner, Klaus Härtel, Jürgen Blumenberg, and Karl Keller. "Combined temperature and pressure testing of thermal protection components." High Temperatures-High Pressures 27/28, no. 3 (1995): 261–66. http://dx.doi.org/10.1068/htrt81.

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Maglić, Kosta, Aleksandar Dobrosavljević, Nenad Perović, Andrej Stanimirović, and Gligo Vuković. "A decade of development and application of millisecond resolution calorimetry between 300 and 2500 K at the Institute of Nuclear Sciences 'Vinca'." High Temperatures-High Pressures 27/28, no. 4 (1995): 389–402. http://dx.doi.org/10.1068/htrt91.

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14

Fujita, Fumitaka, Kunitoshi Uchida, Yasunori Takayama, Yoshiro Suzuki, Masayuki Takaishi, and Makoto Tominaga. "Hypotonicity-induced cell swelling activates TRPA1." Journal of Physiological Sciences 68, no. 4 (June 16, 2017): 431–40. http://dx.doi.org/10.1007/s12576-017-0545-9.

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Abstract Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca2+-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.

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Woo, JooHan, Young Keul Jeon, Yin-Hua Zhang, Joo Hyun Nam, Dong Hoon Shin, and Sung Joon Kim. "Triple arginine residues in the proximal C-terminus of TREK K+ channels are critical for biphasic regulation by phosphatidylinositol 4,5-bisphosphate." American Journal of Physiology-Cell Physiology 316, no. 3 (March 1, 2019): C312—C324. http://dx.doi.org/10.1152/ajpcell.00417.2018.

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TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

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Kunichika, Naomi, Ying Yu, Carmelle V. Remillard, Oleksandr Platoshyn, Shen Zhang, and Jason X. J. Yuan. "Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 5 (November 2004): L962—L969. http://dx.doi.org/10.1152/ajplung.00452.2003.

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Transient receptor potential (TRP) cation channels are a critical pathway for Ca2+ entry during pulmonary artery (PA) smooth muscle contraction. However, whether canonical TRP (TRPC) subunits and which TRP channel isoforms are involved in store depletion-induced pulmonary vasoconstriction in vivo remain unclear. This study was designed to test whether overexpression of the human TRPC1 gene ( hTRPC1) in rat PA enhances pulmonary vasoconstriction due to store depletion-mediated Ca2+ influx. The hTRPC1 was infected into rat PA rings with an adenoviral vector. RT-PCR and Western blot analyses confirmed the mRNA and protein expression of hTRPC1 in the arterial rings. The amplitude of active tension induced by 40 mM K+ (40K) in PA rings infected with an empty adenoviral vector (647 ± 88 mg/mg) was similar to that in PA rings infected with hTRPC1 (703 ± 123 mg/mg, P = 0.3). However, the active tension due to capacitative Ca2+ entry (CCE) induced by cyclopiazonic acid was significantly enhanced in PA rings overexpressing hTRPC1 (91 ± 13% of 40K-induced contraction) compared with rings infected with an empty adenoviral vector (61 ± 14%, P < 0.001). Endothelial expression of hTRPC1 was not involved since the CCE-induced vasoconstriction was also enhanced in endothelium-denuded PA rings infected with the adenoviral vector carrying hTRPC1. These observations demonstrate that hTRPC1 is an important Ca2+-permeable channel that mediates pulmonary vasoconstriction when PA smooth muscle cell intracellular Ca2+ stores are depleted.

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Matsubara, Masaki, Yukiko Muraki, Noriyuki Hatano, Hiroka Suzuki, and Katsuhiko Muraki. "Potent Activation of Human but Not Mouse TRPA1 by JT010." International Journal of Molecular Sciences 23, no. 22 (November 18, 2022): 14297. http://dx.doi.org/10.3390/ijms232214297.

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Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3–100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10–100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.

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BOLLIMUNTHA, SUNITHA, ERIC CORNATZER, and BRIJ B. SINGH. "Plasma membrane localization and function of TRPC1 is dependent on its interaction with β-tubulin in retinal epithelium cells." Visual Neuroscience 22, no. 2 (March 2005): 163–70. http://dx.doi.org/10.1017/s0952523805222058.

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Mammalian homologues of the Drosophila canonical Transient Receptor Potential (TRPC) protein have been proposed to encode the store-operated Ca2+ influx (SOC) channel(s). This study examines the role of TRPC1 in the SOC mechanism of retinal cells. htrpc1 transcript was detected in bovine retinal and in human adult retinal pigment epithelial (ARPE) cells. Western blot analysis also confirmed the expression of TRPC1 protein in neuronal cells including retina and ARPE cells. To determine the role of TRPC1 protein in retinal cells, TRPC1 was recombinantly expressed in ARPE cells and changes in intracellular Ca2+ were analyzed. ARPE cells stably transfected with htrp1 cDNA displayed 2-fold higher Ca2+ influx with no significant increase in the basal influx. Consistent with this the overexpressed TRPC1 protein was localized in the plasma membrane region of ARPE cells. Interestingly, both bovine retinal tissues and ARPE cells showed that TRPC1 protein co-localizes and could be co-immunoprecipitated with β-tubulin. Disruption of tubulin by colchicine significantly decreased both plasma membrane staining of the TRPC1 protein and Ca2+ influx in ARPE cells. These results suggest that TRPC1 channel protein is expressed in retinal cells, further, targeting/retention of the TRPC1 protein to the plasma membrane in retinal cells is mediated via its interaction with β-tubulin.

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Palmaers, Natalie E., Steffen B. Wiegand, Christine Herzog, Frank G. Echtermeyer, Mirjam J. Eberhardt, and Andreas Leffler. "Distinct Mechanisms Account for In Vitro Activation and Sensitization of TRPV1 by the Porphyrin Hemin." International Journal of Molecular Sciences 22, no. 19 (October 8, 2021): 10856. http://dx.doi.org/10.3390/ijms221910856.

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TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.

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Chien, Jeremy, Takayo Ota, Giovanni Aletti, Ravi Shridhar, Mariarosaria Boccellino, Lucio Quagliuolo, Alfonso Baldi, and Viji Shridhar. "Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration." Molecular and Cellular Biology 29, no. 15 (May 26, 2009): 4177–87. http://dx.doi.org/10.1128/mcb.00035-09.

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ABSTRACT HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability.

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Moparthi, Lavanya, Sven Kjellström, Per Kjellbom, Milos R. Filipovic, Peter M. Zygmunt, and Urban Johanson. "Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry." International Journal of Molecular Sciences 21, no. 18 (September 11, 2020): 6667. http://dx.doi.org/10.3390/ijms21186667.

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The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.

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Matsuyama, Minami, Yuko Terada, Toyomi Yamazaki-Ito, and Keisuke Ito. "A Luminescence-Based Human TRPV1 Assay System for Quantifying Pungency in Spicy Foods." Foods 10, no. 1 (January 13, 2021): 151. http://dx.doi.org/10.3390/foods10010151.

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The quantitation of pungency is difficult to achieve using sensory tests because of persistence, accumulation, and desensitization to the perception of pungency. Transient receptor vanilloid 1 (TRPV1), which is a chemosensory receptor, plays a pivotal role in the perception of many pungent compounds, suggesting that the activity of this receptor might be useful as an index for pungency evaluation. Although Ca2+-sensitive fluorescence dyes are commonly used for measuring human TRPV1 (hTRPV1) activity, their application is limited, as foods often contain fluorescent substances that interfere with the fluorescent signals. This study aims to design a new pungency evaluation system using hTRPV1. Instead of employing a fluorescent probe as the Ca2+ indicator, this assay system uses the luminescent protein aequorin. The luminescence assay successfully evaluated the hTRPV1 activity in foods without purification, even for those containing fluorescent substances. The hTRPV1 activity in food samples correlated strongly with the pungency intensity obtained by the human sensory test. This luminescence-based hTRPV1 assay system will be a powerful tool for objectively quantifying the pungency of spicy foods in both laboratory and industrial settings.

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Pan, Shengliu, Min Liu, Huijuan Xu, Junlan Chuan, and Zhenglin Yang. "Lipopolysaccharide Activating NF-kB Signaling by Regulates HTRA1 Expression in Human Retinal Pigment Epithelial Cells." Molecules 28, no. 5 (February 28, 2023): 2236. http://dx.doi.org/10.3390/molecules28052236.

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Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD). However, the specific mechanism that HTRA1 causes AMD and the relationship between HTRA1 and inflammation remains unclear. We found that lipopolysaccharide (LPS) induced inflammation enhanced the expression of HTRA1, NF-κB, and p-p65 in ARPE-19 cells. Overexpression of HTRA1 up-regulated NF-κB expression, and on the other hand knockdown of HTRA1 down-regulated the expression of NF-κB. Moreover, NF-κB siRNA has no significant effect on the expression of HTRA1, suggesting HTRA1 works upstream of NF-κB. These results demonstrated that HTRA1 plays a pivotal role in inflammation, explaining possible mechanism of overexpressed HTRA1-induced AMD. Celastrol, a very common anti-inflammatory and antioxidant drug, was found to suppress inflammation by inhibiting phosphorylation of p65 protein efficaciously in RPE cells, which may be applied to the therapy of age-related macular degeneration.

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Tom, Irene, Victoria C. Pham, Kenneth J. Katschke, Wei Li, Wei-Ching Liang, Johnny Gutierrez, Andrew Ah Young, et al. "Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy." Proceedings of the National Academy of Sciences 117, no. 18 (April 28, 2020): 9952–63. http://dx.doi.org/10.1073/pnas.1917608117.

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Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

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Campbell, Robert A., Heather D. Campbell, J. Samuel Bircher, Claudia Valeria de Araujo, Frederik Denorme, Jacob L. Crandell, John L. Rustad, et al. "Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide." Blood 138, no. 11 (June 30, 2021): 977–88. http://dx.doi.org/10.1182/blood.2020009021.

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Abstract Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. The placenta expresses high-temperature requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1 knockout mice (HTRA1−/−). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1−/− pups had no detectable NIPs, and PMNs from HTRA1−/− pups became NET competent earlier after birth compared with HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.

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Tossetta, Giovanni, Sonia Fantone, Stefano Raffaele Giannubilo, Andrea Ciavattini, Martina Senzacqua, Andrea Frontini, and Daniela Marzioni. "HTRA1 in Placental Cell Models: A Possible Role in Preeclampsia." Current Issues in Molecular Biology 45, no. 5 (May 1, 2023): 3815–28. http://dx.doi.org/10.3390/cimb45050246.

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The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine–protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

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Dallas, Mark L., Jason L. Scragg, and Chris Peers. "Modulation of hTREK-1 by carbon monoxide." NeuroReport 19, no. 3 (February 2008): 345–48. http://dx.doi.org/10.1097/wnr.0b013e3282f51045.

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Catalano, V., A. Baldi, V. Shridhar, M. P. Staccioli, J. Chien, P. Giordani, D. Rossi, et al. "HtrA1 expression as a predictive factor of response to cisplatin-based regimen in patients with advanced gastric cancer." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 4077. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.4077.

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4077 Background: Human HtrA1 is a member of the HtrA (High temperature requirement) family of serine proteases. Recent reports suggest that htrA1 plays a protective role in varous malignancies due to its tumour suppressive properties. This study was performed to estimate HtrA1 expression as a predictor of the response to chemotherapy of patients with gastric cancer. Methods: HtrA1 was measured immunohistochemically on archival specimens of primary gastric cancer from 51 patients treated consecutively at our institution with a weekly chemotherapy including cisplatin 40 mg/m2, epirubicin 35 mg/m2, 6S-leucovorin 100 mg/m2, 5-fluorouracil 500 mg/m2, with the support of filgrastim 5 μg/Kg from the day 2 to 7 (PELF regimen), or cisplatin 40 mg/m2, epirubicin 35 mg/m2, 6S-leucovorin 100 mg/m2, 5-fluorouracil 500 mg/m2 (PLF regimen). Response to chemotherapy was assessed after 8 weekly treatments according to the WHO criteria. Results: our population consisted of M/F 32/19; median age 64 years (range, 46–79). The prevalent metastatic sites were liver (17 pts), peritoneum (13 pts), lymph nodes (21 pts), locoregional disease (16 pts); 31/16/4 pts had 1/2/3 or more sites of disease. 23 pts had a low expression of HtrA1 (0/1+) versus 28 patients with higher expression (2+). Of the total 51 patients, there were 28 responders: 8 showing complete response (CR) and 20, partial response (PR). Of the 28 responders, 20 were in the higher HtrA1 staining group (2+), while of the 23 non-responders, 15 were in the higher HtrA1 staining group (0/1+). A statistically significant correlation between HtrA1 expression (HtrA1 2+ versus HtrA1 0/1+) and the clinical response was observed (response rate in patients with 2+ and 0/1+: 71.4% versus 34.8%, P < 0.01, respectively). Interestingly, among 16 pts with locoregional disease (stomach, gastric bed, anastomosis), 1/6 pts had HtrA1 1+ expression compared to 8/10 pts with HtrA1 2+ (17% versus 80%, respectively; p = 0.025). Conclusions: The immunohistochemical identification of HtrA1 on the primary gastric cancer prior to chemotherapy may be a useful predictor for choice of potentially responders to a cisplatin-based chemotherapy. No significant financial relationships to disclose.

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Vierkotten, Sascha, and Victoria Korinek. "HTRA1 enhance signaling pathway of uveitis via modulation of the TGF-β signaling cascade." American Journal of BioMedicine 4, no. 3 (August 30, 2016): 276–88. http://dx.doi.org/10.18081/2333-5106/016-276-288.

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High temperature requirement protein A1 (HtrA1) is a serine protease that is mostly secreted to degrade numerous extracellular matrix proteins, They are involved in the development and progression of several pathological processes such as cancer, neurodegenerative disorders and arthritic diseases, but it also exists within cells for some partially understood. The purpose of this study was to investigate the role of HTRA1 in the uveitis, and the possible mechanisms involved. Interphotoreceptor retinoid-binding protein peptide R14 to induced uveitis in rat model. A recombinant lentiviral vector carrying HTRA1-shRNA to knockdown HTRA1 expression. ELISA to detected proinflammatory cytokines and standard molecular biological techniques were used for subcloning of HtrA1. The knockdown of HTRA1 was associated with decreased the cellularlevel expression of proinflammatory cytokine resulted in reduced cellular damage, and increased mRNA levels of TGF-β1.

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Supanji, Supanji, Ayudha Bahana Ilham Perdamaian, Anindita Dianratri, Anditta Syifarahmah, Tri Wahyu Widayanti, Firman Setya Wardhana, Muhammad Bayu Sasongko, Mohammad Eko Prayogo, Angela Nurini Agni, and Chio Oka. "HtrA1 serine protease expression levels on age-related macular degeneration (AMD) patients in Yogyakarta." BIO Web of Conferences 28 (2020): 02004. http://dx.doi.org/10.1051/bioconf/20202802004.

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This research aims to investigate the HtrA1 serine protease circulating level of Age-related Macular Degeneration (AMD) patients in Yogyakarta, Indonesia. This study was conducted from January to August 2019 which included 38 AMD patients and 16 Non-AMD patients/controls (two groups). Baseline data and blood sample were collected. ELISA assay was used to measure the HtrA1 serine protease circulating level on both groups. SNP genotyping of rs10490924 was using restriction enzyme digestion. This study used The IBM SPSS® version 24 (Chicago, The USA) to determine the relationship between HtrA1 expression level and AMD incidence. AMD patients had higher HtrA1 serine protease level (35.31) than controls (30.08). However, there is no association found between HtrA1 serine protease level and AMD incidence (p-value>0.05, CI 95 %). However, HtrA1 serine protease did not associate positively to AMD incidence in Yogyakarta samples. Further analysis by grouping AMD patient based on the rs10490924 genotype show no statistical correlation between HTRA1 to the incidence of AMD. This result might be due to the lack of samples in the study groups. Future studies with larger number of samples are advised to better see the association between Htra1 serine protease level and AMD incidence.

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Zurawa-Janicka, Dorota, Jarek Kobiela, Tomasz Slebioda, Rafal Peksa, Marcin Stanislawowski, Piotr Mieczyslaw Wierzbicki, Tomasz Wenta, et al. "Expression of HTRA Genes and Its Association with Microsatellite Instability and Survival of Patients with Colorectal Cancer." International Journal of Molecular Sciences 21, no. 11 (May 31, 2020): 3947. http://dx.doi.org/10.3390/ijms21113947.

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HtrA proteases regulate cellular homeostasis and cell death. Their dysfunctions have been correlated with oncogenesis and response to therapeutic treatment. We investigated the relation between HtrA1-3 expression and clinicopathological, and survival data, as well as the microsatellite status of tumors. Sixty-five colorectal cancer patients were included in the study. The expression of HTRA1-3 was estimated at the mRNA and protein levels by quantitative PCR and immunoblotting. Microsatellite status was determined by high-resolution-melting PCR. We found that the HTRA1 mRNA level was higher in colorectal cancer tissue as compared to the unchanged mucosa, specifically in primary lesions of metastasizing cancer. The levels of HtrA1 and HtrA2 proteins were reduced in tumor tissue when compared to unchanged mucosa, specifically in primary lesions of metastasizing disease. Moreover, a decrease in HTRA1 and HTRA2 transcripts’ levels in cancers with a high level of microsatellite instability compared to microsatellite stable ones has been observed. A low level of HtrA1 or/and HtrA2 in cancer tissue correlated with poorer patient survival. The expression of HTRA1 and HTRA2 changes during colorectal carcinogenesis and microsatellite instability may be, at least partially, associated with these changes. The alterations in the HTRA1/2 genes’ expression are connected with metastatic potential of colorectal cancer and may affect patient survival.

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Lin, Xiaolei, Tianke Yang, Xin Liu, Fan Fan, Xiyue Zhou, Hongzhe Li, and Yi Luo. "TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract." International Journal of Molecular Sciences 23, no. 22 (November 20, 2022): 14431. http://dx.doi.org/10.3390/ijms232214431.

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Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.

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Lindsay, Christopher D., Christopher Green, Mike Bird, James T. A. Jones, James R. Riches, Katherine K. McKee, Mark S. Sandford, Debra A. Wakefield, and Christopher M. Timperley. "Potency of irritation by benzylidenemalononitriles in humans correlates with TRPA1 ion channel activation." Royal Society Open Science 2, no. 1 (January 2015): 140160. http://dx.doi.org/10.1098/rsos.140160.

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We show that the physiological activity of solid aerosolized benzylidenemalononitriles (BMNs) including ‘tear gas’ (CS) in historic human volunteer trials correlates with activation of the human transient receptor potential ankyrin 1 ion channel (hTRPA1). This suggests that the irritation caused by the most potent of these compounds results from activation of this channel. We prepared 50 BMNs and measured their hTRPA1 agonist potencies. A mechanism of action consistent with their physiological activity, involving their dissolution in water on contaminated body surfaces, cell membrane penetration and reversible thiolation by a cysteine residue of hTRPA1, supported by data from nuclear magnetic resonance experiments with a model thiol, explains the structure–activity relationships. The correlation provides evidence that hTRPA1 is a receptor for irritants on nociceptive neurons involved in pain perception; thus, its activation in the eye, nose, mouth and skin would explain the symptoms of lachrymation, sneezing, coughing and stinging, respectively. The structure–activity results and the use of the BMNs as pharmacological tools in future by other researchers may contribute to a better understanding of the TRPA1 channel in humans (and other animals) and help facilitate the discovery of treatments for human diseases involving this receptor.

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Campbell, Robert A., Mark Cody, Yasuhiro Kosaka, Heather D. Campbell, and Christian Yost. "Placental HTRA1 Protease Cleaves Alpha-1-Antitrypsin and Generates Neonatal NET-Inhibitory Factor." Blood 132, Supplement 1 (November 29, 2018): 273. http://dx.doi.org/10.1182/blood-2018-99-111195.

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Abstract BACKGROUND: Neutrophil extracellular traps (NET) are extracellular lattices of decondensed chromatin associated with anti-microbial proteins and degradative enzymes released by polymorphonuclear leukocytes (PMN) to trap and kill invading microbes. Dysregulated NET formation, however, contributes to inflammatory tissue damage. We have identified a novel NET-inhibitory peptide, neonatal NET-Inhibitory Factor (nNIF), present in the fetal circulation. nNIF is formed as a carboxy-terminus cleavage fragment of alpha-1 antitrypsin (AAT), an abundant, circulating protease inhibitor with homologs in human and mouse blood. However, the exact mechanisms by which nNIF is generated in fetal and neonatal blood remains unknown. OBJECTIVE: High temperature requirement A 1 (HTRA1) is expressed in the placenta during fetal development and inhibits AAT. We hypothesized that placentally expressed HTRA1, a serine protease, regulates the formation of NET-inhibitory peptides, such as nNIF, through cleavage of AAT. DESIGN/METHODS: Term and preterm placenta were lysed and probed for HTRA1 expression. HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA. Recombinant, bioactive HTRA1 or placenta-eluted HTRA1 were incubated with AAT and the generation of carboxy-terminus fragments of AAT was assessed using western blotting and mass spectrometry. Fragments of AAT generated by HTRA1 were incubated with LPS-stimulated PMNs and NET formation was examined qualitatively using live cell imaging and quantitatively using a high throughput fluorescence assay. The effect of the HTRA-AAT cleavage fragment on reactive oxygen species generation, neutrophil chemotaxis, phagocytosis, and bacterial killing was measured using flow cytometry, a modified Boyden chamber asssay, neutrophil labeled Escherichia coli uptake assay, and a bacterial killing assay with a pathogenic strain of Escherichia coli, respectively. Finally, NET formation was evaluated qualitatively and quantitatively in murine PMNs isolated from neonatal WT and HTRA1-/- pups between 1-3, 4-6 and 7-10 days after birth to determine when PMNs become NET-competent. RESULTS: Term and preterm infant placentas express HTRA1, and we detected significantly (p<0.05) higher levels of HTRA1 in plasma from term (465.1±71.8 µg/mL) and preterm (385.9±71.3 µg/mL) infant cord blood compared to adults (58.6±11.6 µg/mL). Recombinant, bioactive HTRA1 and placenta-derived HTRA1 incubated with AAT generate a 4kD AAT fragment based on western blot and mass spectrometry similar to the nNIF fragment found in cord blood from term and preterm infants. Pre-incubation of this fragment with LPS-stimulated PMNs significantly inhibits NET formation (p<0.05). The cleavage fragment from HTRA1-AAT, however, has no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly (p<0.05) reduces NET-associated bacterial killing by 62% compared to a scrambled HTRA-AAT control peptide. In addition, the HTRA1-AAT fragment significantly (p<0.05) reduces nuclear decondensation by 93% compared to LPS-stimulated PMN, suggesting this fragment inhibits PAD4 activation similar to other NIFs previously examined. Neonatal murine plasma contains a 4kD AAT fragment which inhibits NET formation by adult mouse PMNs, indicating that nNIF generation is conserved in mice. Neonatal PMNs stimulated with LPS exhibit delayed NET formation following birth with PMNs becoming NET-competent by day 8 of life. However, neonatal PMNs from pups born from HTRA1-/- deficient mice generate significantly (p<0.05) more NETs between day 4-6 of life compared to WT controls, suggesting that HTRA1 regulates NET formation through nNIF production. CONCLUSIONS: Placental HTRA1 interacts with AAT to generate a carboxy-terminus cleavage fragment of AAT with identical NET-inhibitory properties to nNIF. Our data strongly indicate that placental HTRA1 generates nNIF in the fetal circulation. We speculate that nNIF participates in the required tolerance to new microbial antigens encountered during the transition to extrauterine life. Disclosures No relevant conflicts of interest to declare.

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Yoshida, Kanoko, Kazuya Kusama, Yuta Fukushima, Takako Ohmaru-Nakanishi, Kiyoko Kato, and Kazuhiro Tamura. "Alpha-1 Antitrypsin-Induced Endoplasmic Reticulum Stress Promotes Invasion by Extravillous Trophoblasts." International Journal of Molecular Sciences 22, no. 7 (April 1, 2021): 3683. http://dx.doi.org/10.3390/ijms22073683.

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Alpha-1 antitrypsin (A1AT) is a glycoprotein that has been shown to protect tissues from proteolytic damage under various inflammatory conditions. Several studies show that A1AT may be associated with pre-eclampsia. However, the role of A1AT expression in placental physiology is not fully understood. In the present study, we aim to characterize the expression and function of placental A1AT. A1AT knockdown is found to reduce the expression of the serine protease HTRA1 in a trophoblast cell line. In addition, A1AT overexpression (A1AT-OE) increases the expression of HTRA1, IL6, CXCL8, and several markers of endoplasmic reticulum (ER) stress. Treatment with tunicamycin or thapsigargin, which induces ER stress, increases HTRA1 expression. Furthermore, immunohistochemistry reveals that HTRA1 is expressed in trophoblasts and the endometrial decidual cells of human placentas. An invasion assay shows that A1AT and HTRA1 stimulate cell invasion, but treatment with the ER stress inhibitors reduces the expression of HTRA1 and ER stress markers and prevents cell invasion in A1AT-OE trophoblasts. These results suggest that endogenous A1AT regulates inflammatory cytokine expression and HTRA1-induced trophoblast invasion via the induction of ER stress. It is concluded that an imbalance in the functional link between A1AT and ER stress at the maternal–fetal interface might cause abnormal placental development.

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Ahamed, Waseem, Richard Ming Chuan Yu, Yang Pan, Takeshi Iwata, Veluchamy Amutha Barathi, Yeo Sia Wey, Sai Bo Bo Tun, et al. "HTRA1 Regulates Subclinical Inflammation and Activates Proangiogenic Response in the Retina and Choroid." International Journal of Molecular Sciences 23, no. 18 (September 6, 2022): 10206. http://dx.doi.org/10.3390/ijms231810206.

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High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg—33% (20/60) and wild-type (WT)—7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.

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Canfield, A. E., K. D. Hadfield, C. F. Rock, E. C. Wylie, and F. L. Wilkinson. "HtrA1: a novel regulator of physiological and pathological matrix mineralization?" Biochemical Society Transactions 35, no. 4 (July 20, 2007): 669–71. http://dx.doi.org/10.1042/bst0350669.

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HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-β (transforming growth factor-β)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-β/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.

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Zurawa-Janicka, Dorota, Jaroslaw Kobiela, Tomasz Stefaniak, Agnieszka Wozniak, Joanna Narkiewicz, Michał Wozniak, Janusz Limon, and Barbara Lipinska. "Changes in expression of serine proteases HtrA1 and HtrA2 during estrogen-induced oxidative stress and nephrocarcinogenesis in male Syrian hamster." Acta Biochimica Polonica 55, no. 1 (January 30, 2008): 9–20. http://dx.doi.org/10.18388/abp.2008_3123.

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Serine proteases HtrA1 and HtrA2 are involved in cellular stress response and development of several diseases, including cancer. Our aim was to examine the involvement of the HtrA proteins in acute oxidative stress response induced in hamster kidney by estrogen treatment, and in nephrocarcinogenesis caused by prolonged estrogenization of male Syrian hamster. We used semi-quantitative RT-PCR to estimate the HtrA1 and HtrA2 mRNA levels in kidney tissues, and Western blotting to monitor the amount of the HtrA proteins. Within the first five hours following estrogen administration both HtrA1 mRNA and the protein levels were increased significantly. No changes in the expression of HtrA2 were observed. This indicates that HtrA1 may be involved in the response against oxidative stress induced by estrogen treatment in hamster kidney. During prolonged estrogenization, a significant reduction of the HtrA1 mRNA and protein levels was observed after 6 months of estradiol treatment, while the expression of HtrA2 was significantly elevated starting from the third month. This suggests an involvement of the HtrA proteins in estrogen-induced nephrocarcinogenesis in hamster. Using fluorescence in situ hybridization we localized the HtrA1 gene at the qb3-4 region of Syrian hamster chromosome 2, the region known to undergo a nonrandom deletion upon prolonged estrogenization. It is possible that the reduced level of HtrA1 expression is due to this chromosomal aberration. A full-length cDNA sequence of the hamster HtrA1 gene was obtained. It codes for a 50 kDa protein which has 98 and 96% identity with mouse and human counterparts, respectively.

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Tossetta, Giovanni, Sonia Fantone, Rosaria Gesuita, Gian Carlo Di Renzo, Arun Meyyazhagan, Chiara Tersigni, Giovanni Scambia, Nicoletta Di Simone, and Daniela Marzioni. "HtrA1 in Gestational Diabetes Mellitus: A Possible Biomarker?" Diagnostics 12, no. 11 (November 5, 2022): 2705. http://dx.doi.org/10.3390/diagnostics12112705.

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Background: The high-temperature requirement A 1 (HtrA1) is a multidomain secretory protein with serine-protease activity, expressed in many tissues, including placenta, where its expression is higher in the first trimester, suggesting an association of this serine protease in early phases of human placenta development. In this study, we evaluated maternal serum HtrA1 levels in the first and third trimester of gestation. In particular, we evaluated a possible role of HtrA1 as an early marker of gestational diabetes mellitus (GDM) in the first trimester of gestation. Methods: We evaluated HtrA1 serum levels in the third trimester (36–40 weeks) in normal pregnancies (n = 20) and GDM pregnancies (n = 20) by using ELISA analysis. Secondly, we performed the same analysis by using the first trimester sera (10–12 weeks) of healthy pregnant women that will develop a normal pregnancy (n = 210) or GDM (n = 28) during pregnancy. Results: We found that HtrA1 serum levels in the third trimester were higher in pregnancies complicated by GDM. Interestingly, higher HtrA1 serum levels were also found in the first trimester in women developing GDM later during the second–third trimester. No significant differences in terms of maternal age and gestational age were found between cases and controls. Women with GDM shown significantly higher pre-pregnancy BMI values compared to controls. Moreover, the probability of GDM occurrence significantly increased with increasing HtrA1 levels and BMI values. The ROC curve showed a good accuracy in predicting GDM, with an AUC of 0.74 (95%CI: 0.64–0.92). Conclusions: These results suggest an important role of HtrA1 as an early predictive marker of GDM in the first trimester of gestation, showing a significative clinical relevance for prevention of this disease.

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Bowden, M. A., L. A. Di Nezza, T. Jobling, L. A. Salamonsen, and G. Nie. "284.Expression of HtrA1, 2 and 3 in human endometrial cancer." Reproduction, Fertility and Development 16, no. 9 (2004): 284. http://dx.doi.org/10.1071/srb04abs284.

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The mammalian HtrA family consists of serine proteases with distinct domains homologous to the bacterial high temperature requirement factor (HtrA). Three human HtrA members have been reported: HtrA1 (PRSS11 or L56), HtrA2 (OMI) and HtrA3 (PRSP). The function of HtrA1 is not well characterised, but it has been shown to be downregulated in malignant tissues (1–3) indicating that the downregulation of HtrA1 is associated with cancer progression. HtrA2 regulates apoptosis by interacting with X-linked inhibitors of apoptosis (XIAP) thus preventing the caspase-inhibitory function of XIAP (4). The function of newly identified HtrA3 is not known, however it shares a high degree of sequence and domain homologies with HtrA1 and may therefore share a functional similarity with HtrA1 (5). Endometrial cancer (EC) is a prevalent gynaecological cancer, commonly affecting women after menopause. In this study we examined the expression of HtrA1, 2 and 3 in EC. Reverse transcriptase-PCR (semi-quantitative) analysis showed decreased mRNA expression of both HtrA1 and HtrA3, but no significant change for HtrA2, in EC tissue samples compared to normal endometrium. We then determined the protein level of expression and the cellular localisation of all three HtrA members in EC progression using immunohistochemistry. HtrA1 and HtrA3 showed a similar pattern of expression and both decreased dramatically with the progression of cancer from grade 1 through to 3. Surprisingly, HtrA2 protein expression was also decreased with cancer progression, but the decline was not as dramatic as that for HtrA1 and HtrA3. Interestingly, considerably less staining was observed for all three HtrA proteins in grade 3 cancer tissues. These data suggest that decreased expression of HtrA proteins, particularly HtrA1 and HtrA3, is associated with the progression of endometrial cancer. (1) Nie, G., Hampton, A., Li, Y., Findlay, J., Salamonsen, L.A. (2003) Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2. Biochem. J. 371, 39–48. (2) van Loo, G., van Gurp, M., Depuydt, B., Srinivasula, S.M., Rodriguez, I., Alnemri, E.S., Gevaert, K., Vandekerckhove, J., Declercq, W., Vandenabeele, P. (2002) The serine protease OMI/HtrA2 is released from mitochondria during apoptosis. OMI interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Diff. 9, 20–26. (3) Chien, J., Staub, J., Hu, S., Erickson-Johnson, M.R., Couch, F.J., Smith, D.I., Crowl, R.M., Kaufmann, S., Shridhar, V. (2004) A candidate tumour supressor HtrA1 is down-regulated in ovarian cancer. Oncogene 23, 1636–1644. (4) Shridhar, V., Sen, A., Chien, J., Staub, J., Avula, R., Kovats, S., Lee, J., Lillie, J., Smith, D.I. (2002) Identification of underexpressed genes in early- and late-stage primary ovarian tumours by suppression subtraction hybridization. Cancer Res. 62, 262–270. (5) Baldi, A., De Luca, A., Morini, M., Battista, T., Felsani, A., Baldi, F., Catricala, C., Amantea, A., Noonan, D. M., Albini, A., Ciorgio, P., Lombardi, D., Paggi, M. G. (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21, 6684–6688.

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Al-Rabadi, Laith Farah, Tiffany Caza, Claire Trivin-Avillach, Aylin R. Rodan, Nicole Andeen, Norifumi Hayashi, Brandi Williams, et al. "Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy." Journal of the American Society of Nephrology 32, no. 7 (May 5, 2021): 1666–81. http://dx.doi.org/10.1681/asn.2020101395.

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BackgroundIdentification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%–20% of patients.MethodsA multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray.ResultsThese approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 “quadruple-negative” (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%.ConclusionsConventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.

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Bougea, Anastasia, George Velonakis, Nikolaos Spantideas, Evangelos Anagnostou, George Paraskevas, Elisabeth Kapaki, and Evangelia Kararizou. "The first Greek case of heterozygous cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy: An atypical clinico-radiological presentation." Neuroradiology Journal 30, no. 6 (April 12, 2017): 583–85. http://dx.doi.org/10.1177/1971400917700168.

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Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) was previously considered a rare, early-onset recessive form of small-vessel disease (SVD) caused by biallelic mutations in the serine protease gene HTRA1 with subsequent loss of its activity. However, very recently, there is growing interest of research showing heterozygous HTRA1 mutations as causes of SVD with a dominant inheritance pattern. This first Greek heterozygous CARASIL case with unusual clinico-radiological presentation extends our very recent knowledge on how heterozygous CARASIL mutations may be associated with cerebral SVD. Our findings highlight heterozygous HTRA1 mutations as an important cause of familial SVD, and that screening of HTRA1 should be considered in all patients with a hereditary SVD of unknown aetiology.

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Ibrahimi, Muhammad, Hiroaki Nozaki, Angelica Lee, Osamu Onodera, Raymond Reichwein, Matthew Wicklund, and Mohammad El-Ghanem. "A CARASIL Patient from Americas with Novel Mutation and Atypical Features: Case Presentation and Literature Review." Cerebrovascular Diseases 44, no. 3-4 (2017): 135–40. http://dx.doi.org/10.1159/000477358.

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Objective: Reporting a novel mutation in the HTRA1 gene in a CARASIL patient from Americas. Methods: Clinical presentation and neuroimaging were consistent with CARASIL. HTRA1 DNA sequencing was performed using advanced (“next generation”) sequencing technology. The results revealed a homozygous missense mutation as c.616G>A (p.Gly206Arg) in the HTRA1 gene. Results: A 24-year-old man with a history of chronic back pain presented with recurrent ischemic strokes. A diagnosis of CARASIL was made with the finding of a novel homozygous missense mutation c.616G>A in HTRA1 gene, resulting in change from Glycine to Arginine in the Serine Protease HTRA1. Brain imaging showed multiple lacunar infarcts with extensive abnormalities of the white matter that spared the external capsules. He also had unilateral decreased hearing with craniofacial asymmetry. None of the above features have been previously described in known CARASIL patients. Both parents of the proband were heterozygous for the same missense mutation. Conclusion: We discovered a novel missense mutation (c.616G>A) associated with a phenotype of CARASIL. This is the first genetically backed case of CARASIL in the new world. The patient's craniofacial abnormalities, including asymmetry of the head, may be related to impaired modulation of transforming growth factor-β1, the result of loss of proteolytic activity of HTRA1. External capsules remained unaffected, despite findings of advanced changes in the rest of the cerebral white matter. Literature is briefly reviewed. The patient's history, neurological exam, neuroimaging, and genetic testing are included.

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BROWNLOW, Sharon L., and Stewart O. SAGE. "Rapid agonist-evoked coupling of type II Ins(1,4,5)P3 receptor with human transient receptor potential (hTRPC1) channels in human platelets." Biochemical Journal 375, no. 3 (November 1, 2003): 697–704. http://dx.doi.org/10.1042/bj20030929.

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Depletion of intracellular Ca2+ stores results in the activation of SMCE (store-mediated Ca2+ entry) in many cells. The mechanism of activation of SMCE is poorly understood. In human platelets, a secretion-like coupling model may be involved. This proposes that store depletion results in trafficking of portions of the endoplasmic reticulum to the plasma membrane, enabling coupling between proteins in the two membranes. In support of this, we have shown that, in human platelets, agonist-evoked Ca2+ store depletion results in de novo and reversible coupling of the InsP3RII [type II inositol (1,4,5)trisphosphate receptor] with the putative Ca2+ entry channel hTRPC1 [human canonical transient receptor potential 1 (protein); Rosado, Brownlow and Sage (2002) J. Biol. Chem. 277, 42157–42163]. A crucial test of the hypothesis that this coupling activates SMCE is that it should occur rapidly enough to account for agonist-evoked Ca2+ entry. In the present study, we have used quenched- and stopped-flow approaches to determine the latencies of thrombin-evoked coupling of InsP3RII with hTRPC1 and of thrombin-evoked bivalent cation entry using Mn2+ quenching of fura 2 fluorescence. Thrombin-evoked Mn2+ entry was detected with a latency of 0.81±0.07 s (S.E.M., n=7) or 1.36±0.09 s (S.E.M., n=7) at a concentration of 1.0 or 0.1 unit/ml respectively. Coupling between InsP3RII and hTRPC1, assessed at 100 ms intervals, was first detected with a latency of 0.9 or 1.4 s after stimulation with thrombin at a concentration of 1.0 or 0.1 unit/ml respectively. These results support the hypothesis that de novo coupling of InsP3RII with hTRPC1 could activate SMCE in human platelets.

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Ciferri, Claudio, Michael T. Lipari, Wei-Ching Liang, Alberto Estevez, Julie Hang, Scott Stawicki, Yan Wu, et al. "The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody." Biochemical Journal 472, no. 2 (November 13, 2015): 169–81. http://dx.doi.org/10.1042/bj20150601.

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The high temperature requirement A1 (HtrA1) protease is implicated in many pathological processes, including the age-related macular degeneration (AMD). We identified a blocking antibody binding to the HTRA1 complex trimer and were able to elucidate an unusual inhibitory mechanism by structural and biochemical experiments.

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Williams, Brandi L., Nathan A. Seager, Jamie D. Gardiner, Chris M. Pappas, Monica C. Cronin, Cristina Amat di San Filippo, Robert A. Anstadt, et al. "Chromosome 10q26–driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium." Proceedings of the National Academy of Sciences 118, no. 30 (July 22, 2021): e2103617118. http://dx.doi.org/10.1073/pnas.2103617118.

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Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151–171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389–407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227–3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE–Bruch’s membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE–BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

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Mukherjee, Sourajit, and Sujit Sikdar. "Polymodal sensitivity of hTREK-1 channel to ischemia related factors." IBRO Reports 6 (September 2019): S361. http://dx.doi.org/10.1016/j.ibror.2019.07.1147.

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Taule-Sivertsen, Peter, Ove Bruland, Aril Løge Håvik, Eirik Bratland, Morten Lund-Johansen, and Per Morten Knappskog. "The SH3PXD2A-HTRA1 fusion transcript is extremely rare in Norwegian sporadic vestibular schwannoma patients." Journal of Neuro-Oncology 154, no. 1 (July 2, 2021): 35–40. http://dx.doi.org/10.1007/s11060-021-03796-6.

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Abstract Introduction Vestibular schwannoma (VS) is a benign intracranial tumor in which the underlying genetics is largely uncertain, apart from mutations in the tumor suppressor gene NF2. Alternative tumorigenic mechanisms have been proposed, including a recurrent in-frame fusion transcript of the HTRA1 and SH3PXD2A genes. The gene product of the SH3PXD2A-HTRA1 fusion has been shown to promote proliferation, invasion and resistance to cell death in vitro and tumor growth in vivo. The aim of this study was to replicate the findings and to investigate the frequency of this fusion gene in another cohort of vestibular schwannoma patients. Methods The SH3PXD2A-HTRA1 transcript was synthesized in vitro using PCR and used as a positive control to assess the sensitivity of a real-time PCR assay. This real-time PCR assay was used to search for the presence of the fusion transcript in 121 Norwegian sporadic VS patients. Results The real-time PCR assay showed a high sensitivity and was able to detect as low as ~ 5 copies of the fusion transcript. Out of the 121 investigated tumors, only 1 harbored the SH3PXD2A-HTRA1 fusion. Conclusion Even though the SH3PXD2A-HTRA1 fusion has been shown to be a driver of tumorigenesis, our results suggest that it is a rare event in our VS patients. Further investigation is warranted in order to elucidate whether our results represent an extreme, and if the fusion is present also in other neoplasms.

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Massart, Catherine, Rémy Sapin, Jacqueline Gibassier, Arnaud Agin, and Michèle d'Herbomez. "Intermethod Variability in TSH-Receptor Antibody Measurement: Implication for the Diagnosis of Graves Disease and for the Follow-Up of Graves Ophthalmopathy." Clinical Chemistry 55, no. 1 (January 1, 2009): 183–86. http://dx.doi.org/10.1373/clinchem.2008.115162.

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Abstract Background: We compared the analytical and clinical performance of 3 porcine thyroid receptor antibody (TRAb) methods (1 second- and 2 new third-generation systems) with the conventional TRAb assay based on the human recombinant TSH receptor (hTRAK). Patients and Methods: We obtained sera from 86 patients with untreated Graves disease (GD) and 71 healthy controls. We measured TRAb concentrations by radioreceptor assay using the hTRAK (Brahms) or the porcine TSH receptor (pRRA) from Beckman-Coulter, by electrochemiluminescence immunoassay (ECLIA) with the Elecsys/Cobas (Roche), and by ELISA using the Medizym TRAb clone (Medipan). Results: Between-run assay imprecision was ≤10% and ≤7.6% for hTRAK and ECLIA, but reached 14% and 14.9% for ELISA and pRRA, respectively. Maximal specificity and sensitivity close to 100% were obtained for hTRAK, ECLIA, and ELISA. pRRA failed to detect positive TRAbs in 5 GD patients. Although calibrated against the same reference standard 90/672, the assays displayed a high intermethod variability. The results were significantly higher by ECLIA and lower by ELISA and pRRA compared with hTRAK. Patients with ophthalmopathy had higher TRAb results by ELISA and pRRA than those without eye disease. Conclusions: Second- and third-generation TRAb assays had similar diagnostic sensitivities in the diagnostic evaluation of GD. Despite the use of the same reference standard for calibration, high intermethod variability in TRAb assay results was seen in untreated GD patients. Assay harmonization is necessary for correct interpretation in the follow-up of Graves ophthalmopathy.

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REDONDO, P. C., A. G. S. HARPER, M. T. HARPER, S. L. BROWNLOW, J. A. ROSADO, and S. O. SAGE. "hTRPC1-associated α-actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation." Journal of Thrombosis and Haemostasis 5, no. 12 (December 2007): 2476–83. http://dx.doi.org/10.1111/j.1538-7836.2007.02773.x.

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Journal articles on the topic 'HTREK1'

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