Chloride channel accessory 1 integrates chloride channel activity and mTORC1 in aging‐related kidney injury

The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging‐related kidney injury, we performed RNA‐Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H₂S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H₂S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl⁻ current via the Ca⁺⁺‐dependent Cl⁻ channel TMEM16A (anoctamin‐1) by patch‐clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence‐associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1‐TMEM16A‐Cl⁻ current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H₂S.     

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC₅₀ = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of β-escin-permeabilized ileum SM at constant pCa 6.3 (EC₅₀ = 2 μm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697–880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca²⁺ sensitization of smooth muscle force.The contractile state of smooth muscle (SM)³ is driven by phosphorylation of the regulatory myosin light chain and reflects the balance of the Ca²⁺-calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP) activities (1). The stoichiometry between force and [Ca²⁺] varies with different agonists (2), reflecting other signaling pathways that modulate the MLCP or myosin light chain kinase activities (3–5). Agonist activation of G-protein-coupled receptors triggers Ca²⁺ release from the sarcoplasmic reticulum. Simultaneously, G-protein-coupled receptor signals are mediated by Ca²⁺-independent phospholipase A2 (6) and initiate kinase signals, such as PKC, phosphoinositide 3-kinase (7), and ROCK. These lead to inhibition of MLCP activity resulting in an increase in regulatory myosin light chain phosphorylation independent of a change in Ca²⁺ (Ca²⁺ sensitization) (for review, see Ref. 1). K⁺ depolarization can also activate RhoA in a Ca²⁺-dependent manner (8). Conversely, Ca²⁺ desensitization occurs when nitric oxide production and the activation of Gas elevate cGMP and cAMP levels in SM, leading to dis-inhibition and restoration of MLCP activity (9–15). Thus, MLCP plays a pivotal role in controlling phosphorylation of myosin, in response to physiological stimulation.MLCP is a trimeric holoenzyme consisting of a catalytic subunit of protein phosphatase 1 (PP1) δ isoform and a regulatory complex of MYPT1 and an accessory M21 subunit (16). A PP1 binding site, KVKF³⁸, is located at the N terminus of MYPT1 followed by an ankyrin-repeat domain. This N-terminal domain forms a part of the active site together with the catalytic subunit and controls the substrate specificity via allosteric interaction and targeting to loci (17). The C-terminal region of MYPT1 directly binds to substrates such as myosin and ezrin/radixin/moecin proteins as well as, under some conditions, the plasma membrane, tethering the catalytic subunit to multiple targets (18, 19). Furthermore, MYPT1 is involved in the regulation of MLCP activity. Alternative splicing of MYPT1 occurs in SM depending on the tissue and the developmental stage (20). An exon 13 splicing of MYPT1 is involved in Ca²⁺ sensitization that occurs in response to GTP (21), whereas a splice variant of MYPT1, containing the C-terminal Leu-zipper sequence, correlates with cGMP-dependent relaxation of smooth muscle (22). Direct binding of PKG to MYPT1 at the Leu-zipper domain and/or Arg/Lys-rich domain is involved in the activation of MLCP (23–25). In addition, a myosin phosphatase-Rho interacting protein (M-RIP) is directly associated with the MYPT1 C-terminal domain, proposed to recruit RhoA to the MLCP complex (26). The C-terminal region also binds to ZIP kinase, which phosphorylates MYPT1 at Thr-696⁴ (27). Thus, the C-terminal domain of MYPT1 functions as a scaffold for multiple phosphatase regulatory proteins.Phosphorylation of MYPT1 at Thr-696 and Thr-853 and the phosphatase inhibitory protein CPI-17 at Thr-38 play dominant roles in the agonist-induced inhibition of MLCP (18, 28–34), yet the molecular mechanism(s) of MYPT1 inhibitory phosphorylation is poorly understood. Receptor activation induces biphasic contraction of SM, reflecting a sequential activation of PKC and ROCK. Phosphorylation of CPI-17 occurs first in parallel with Ca²⁺ release and the activation of a conventional PKC that causes Ca²⁺-dependent Ca²⁺ sensitization (35). A delayed activation of ROCK increases the phosphorylation of MYPT1 at Thr-853. These phosphorylation events maintain the sustained phase of contraction after the fall in [Ca²⁺]_i (35). Phosphorylation of MYPT1 at Thr-853 is elevated in response to various agonists (35, 36). Unlike the Thr-853 site, phosphorylation of MYPT1 at Thr-696 is often spontaneously phosphorylated under resting conditions and insensitive to stimuli with most agonists (36). Nonetheless, up-regulation of MYPT1 phosphorylation at Thr-696 is reported in some types of hypertensive animals and patients, suggesting an importance of the site under pathological conditions (37–39). Phosphorylation of CPI-17 and MYPT1 at Thr-696 is reversed in response to nitric oxide production and cGMP elevation, which parallels relaxation (14, 15). Upon cGMP elevation, MYPT1 at Ser-695 is phosphorylated, and the Ser phosphorylation blocks the adjacent phosphorylation at Thr-696, causing dis-inhibition of MLCP (27, 40). However, Ser-695 phosphorylation does not cause the dephosphorylation at Thr-696 in intact cerebral artery (41). Thus, phosphorylation of MYPT1 governs Ca²⁺ sensitization and desensitization of SM, although the underlying mechanisms are still controversial. In addition, telokin, a dominant protein in visceral and phasic vascular SM tissues, is phosphorylated by PKG and PKA, activating MLCP by an unknown mechanism and inducing SM relaxation (42).Multiple mechanisms have been suggested for the phosphorylation-dependent inhibition of MLCP. Thiophosphorylation of MYPT1 results in lower V_m and higher K_m values of MLCP activity, suggesting that allosteric modulation of the active site is necessary for the thiophosphorylation-dependent inhibition of MLCP (43). On the other hand, translocation of MYPT1 to the plasma membrane region occurs in parallel with the phosphorylation of MYPT1 at Thr-696 (44, 45), but the amount translocated and the functional meaning remain controversial (41). Phosphorylation of MYPT1 at Thr-853 in vitro reduces its affinity for phospho-myosin, thus suppressing the phosphatase activity (18). It has also been demonstrated that reconstitution of thiophosphorylated MYPT1 at Thr-696 or Thr-853 with isolated PP1δ produces a less-active form of MLCP complex (46). This supports the kinetic analysis (43) that suggests an allosteric effect of MYPT1 phosphorylation on the phosphatase activity. In contrast, a thiophosphopeptide mimicking the phosphorylation site of MBS85, a homolog of MYPT1 and not present in SM, inhibits the activity of MBS85·PP1 complex, suggesting the direct interaction between the MBS85 site and PP1 (47). In the crystal structure model of MYPT1-(1–229). PP1δ complex, the electrostatic potential map at the MLCP active site complements amino acid profiles around the phosphorylation sites (17). Therefore, it is possible that the inhibitory phosphorylation sites directly dock at the active site of MLCP and inhibit the activity. Here, we examine mechanisms underlying the inhibition of MLCP through the phosphorylation of MYPT1 at Thr-696 and Thr-853 using GST fusion versions of various MYPT1 fragments including or excluding either or both of these phosphorylation sites. Phosphorylated MYPT1 fragments including either Thr-696 or Thr-853 potently and specifically inhibit MLCP purified from pig aorta and the enzyme associated with myofilaments in permeabilized ileum SM tissues. We further show that inhibition of MLCP in SM tissues is eliminated by activation of PKA/PKG, suggesting that the GST-MYPT1 fragments mimic agonist-induced autoinhibition and cAMP/cGMP-dependent dis-autoinhibition of MLCP in SM.     

Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson’s disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16–17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16–24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.     

Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3

This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie. In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm. Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain. Experiments on larval survivability and viable cell count revealed peak immune response on third day. Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set. The haemolymph of A. assama larvae was analyzed for proteins associated with bacterial infection. Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae. Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae. Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts. General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.     

Assessing Essential Trace Elements in Cave Nectar Bat (Eonycteris spelaea): A Study in Barak Valley of Assam,India

Biological Trace Element Research - This study investigated trace elements in the different organs of Eonycteris spelaea, a hill cave from the Bhuban Hills of Sonai Reserve Forest, Cachar, Assam...     

Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum

Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.