NH2-Terminal Cleavage of Cardiac Troponin I Signals Adaptive Response to Cardiac Stressors.
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ABSTRACT:
SUBMITTER: Warren CM
PROVIDER: S-EPMC8444995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF-hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an "open" conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148-158] for cNTnC and cTnI[39-60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1-73] in complex with cTnC. cTnI[39-60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41-67] and a type VIII turn in cTnI[38-41]. In contrast, cTnI[1-37] remains disordered, although cTnI[19-37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35-41]. Domain positioning impacts the effective concentration of cTnI[148-158] presented to cNTnC, and this is how cTnI[19-37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank-Starling law of the heart.
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Project description:Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1-167) and cTnI(1-193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 ?m) and long (2.2 ?m) sarcomere lengths (SLs). Our results show that the cTnI(1-167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1-167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.
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Project description:Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca(2+). We have determined the crystal structure of cTnC, with three bound Ca(2+) ions, complexed with the calcium-sensitizer bepridil, to 2.15-A resolution. In contrast to apo- and 3Ca(2+)-cTnC, the drug-bound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca(2+)-sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147-163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca(2+)-sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca(2+) regulation by troponin C in cardiac muscle.
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Project description:The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH2 -terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH2 -terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH2 -terminal acetylation on O2 binding properties. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid Basic Protocol 2: Selection of E. coli expression strains for coexpression Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification Support Protocol 1: Measuring O2 equilibration curves Support Protocol 2: Mass spectrometry to confirm NH2 -terminal acetylation.
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Project description:Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ∼32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1-73 residues with the corresponding 1-41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca(2+) sensitivity (pCa(50)) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μm. Ca(2+) sensitivity increased significantly in TG fibers at both short SL (pCa(50) of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa(50) of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca(2+). Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function.
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Project description:Autophagy is required for cellular homeostasis and can determine cell viability in response to stress. It is established that MTOR is a master regulator of starvation-induced macroautophagy/autophagy, but recent studies have also implicated an essential role for the MAPK8/cJun NH2-terminal kinase 1 signal transduction pathway. We found that MAPK8/JNK1 and MAPK9/JNK2 were not required for autophagy caused by starvation or MTOR inhibition in murine fibroblasts and epithelial cells. These data demonstrate that MAPK8/9 has no required role in starvation-induced autophagy. We conclude that the role of MAPK8/9 in autophagy may be context-dependent and more complex than previously considered.AbbreviationsAKT: thymoma viral proto-oncogene;ALB: albumin; ATG4: autophagy related 4; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CQ: chloroquine diphosphate; DMEM: Dulbecco's modified Eagle's medium; EDTA: ethylenediaminetetraacetic acid; EBSS: Earle's balanced salt solution; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HRAS: Harvey rat sarcoma virus oncogene; IgG: Immunoglobulin G; MAPK3/ERK1: mitogen-activated protein kinase 3; MAPK8/JNK1: mitogen-activated protein kinase 8; MAPK9/JNK2: mitogen-activated protein kinase 9; MAPK10/JNK3: mitogen-activated protein kinase 10; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; RPS6KB1/p70: ribosomal protein S6 kinase, polypeptide 1; PPARA: peroxisome proliferator activated receptor alpha; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; TRP53: transforming related protein 53; TUBA: tubulin alpha; UV: ultraviolet; WT: wild-type.
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| S-EPMC6143545 | biostudies-literature
Project description:Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.
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Project description:Our previous studies (Howarth, J. W., Meller, J., Solaro, R. J., Trewhella, J., and Rosevear, P. R. (2007) J. Mol. Biol. 373, 706-722) of the unique N-terminal region of human cardiac troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional cross-linker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intra- and intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca(2+)-dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled troponin. Ca(2+) regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca(2+), but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca(2+). This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra- and intermolecular sites.
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