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991.
Justin F. Shaffer Robert W. Kensler Samantha P. Harris 《The Journal of biological chemistry》2009,284(18):12318-12327
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein
expressed in cardiac sarcomeres that is known to interact with myosin, titin,
and actin. cMyBP-C modulates actomyosin interactions in a
phosphorylation-dependent way, but it is unclear whether interactions with
myosin, titin, or actin are required for these effects. Here we show using
cosedimentation binding assays, that the 4 N-terminal domains of murine
cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation
constant (Kd) of ∼10 μm and a molar
binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol)
binding to actin. Electron microscopy and light scattering analyses show that
these domains cross-link F-actin filaments, implying multiple sites of
interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or
m-domain, reduced binding to actin (reduced Bmax) and
eliminated actin cross-linking. These results suggest that the N terminus of
cMyBP-C interacts with F-actin through multiple distinct binding sites and
that binding at one or more sites is reduced by phosphorylation. Reversible
interactions with actin could contribute to effects of cMyBP-C to increase
cross-bridge cycling.Cardiac myosin-binding protein C
(cMyBP-C)2
is a thick filament accessory protein that performs both structural and
regulatory functions within vertebrate sarcomeres. Both roles are likely to be
essential in deciphering how a growing number of mutations found in the
cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure
in a substantial number of the world''s population
(1,
2).Considerable progress has recently been made in determining the regulatory
functions of cMyBP-C and it is now apparent that cMyBP-C normally limits
cross-bridge cycling kinetics and is critical for cardiac function
(3-5).
Phosphorylation of cMyBP-C is essential for its regulatory effects because
elimination of phosphorylation sites (serine to alanine substitutions)
abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge
cycling kinetics and blunts cardiac responses to inotropic stimuli
(6). The substitutions further
impair cardiac function, reduce contractile reserve, and cause cardiac
hypertrophy in transgenic mice
(6,
7). By contrast, substitution
of aspartic acids at these sites to mimic constitutive phosphorylation is
benign or cardioprotective
(8).Although a role for cMyBP-C in modulating cross-bridge kinetics is
supported by several transgenic and knock-out mouse models
(6,
7,
9,
10), the precise mechanisms by
which cMyBP-C exerts these effects are not completely understood. For
instance, the unique regulatory motif or “m-domain” of cMyBP-C
binds to the S2 subfragment of myosin in vitro
(11) and binding is abolished
by PKA-mediated phosphorylation of the m-domain
(12). These observations have
led to the idea that (un)binding of the m-domain from myosin S2 mediates
PKA-induced increases in cross-bridge cycling kinetics. Consistent with this
idea, Calaghan and colleagues
(13) showed that S2 added to
transiently permeabilized myocytes increased their contractility, presumably
because added S2 displaced cMyBP-C from binding endogenous S2. However, other
reports indicate that cMyBP-C can influence actomyosin interactions through
mechanisms unrelated to S2 binding, because either purified cMyBP-C
(14) or recombinant N-terminal
domains of cMyBP-C (15)
affected acto-S1 filament sliding velocities and ATPase rates in the absence
of myosin S2. These results thus raise the possibility that interactions with
ligands other than myosin S2, such as actin or myosin S1, contribute to
effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge
cycling kinetics is supported by several studies that implicate the regulatory
m-domain or sequences near it in actin binding
(16-19).
cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and
consists of 11 repeating domains that bear homology to either Ig or
fibronectin-like folds. Domains are numbered sequentially from the N terminus
of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100
amino acids, is located between domains C1 and C2 and is phosphorylated on at
least 3 serine residues by PKA
(12). Although the precise
structure of the m-domain is not known, small angle x-ray scattering data
suggest that it is compact and folded in solution and is thus similar in size
and dimensions to the surrounding Ig domains
(20). Recombinant proteins
encompassing the m-domain and/or a combination of adjacent domains including
C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been
shown to bind actin (16,
18,
19).The purpose of the present study was to characterize binding interactions
of the N terminus of cMyBP-C with actin and to determine whether interactions
with actin are influenced by phosphorylation of the m-domain. Results
demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin
filaments with affinities similar to that reported for cMyBP-C binding to
myosin S2 (11). Furthermore,
actin binding was reduced by m-domain phosphorylation, suggesting that
reversible interactions of cMyBP-C with actin could contribute to modulation
of cross-bridge kinetics. 相似文献
992.
Meghan E. McGee-Lawrence Samantha J. Wojda Lindsay N. Barlow Thomas D. Drummer Kevin Bunnell Janene Auger Hal L. Black Seth W. Donahue 《Journal of biomechanics》2009,42(10):1378-1383
Disuse typically uncouples bone formation from resorption, leading to bone loss which compromises bone mechanical properties and increases the risk of bone fracture. Previous studies suggest that bears can prevent bone loss during long periods of disuse (hibernation), but small sample sizes have limited the conclusions that can be drawn regarding the effects of hibernation on bone structure and strength in bears. Here we quantified the effects of hibernation on structural, mineral, and mechanical properties of black bear (Ursus americanus) cortical bone by studying femurs from large groups of male and female bears (with wide age ranges) killed during pre-hibernation (fall) and post-hibernation (spring) periods. Bone properties that are affected by body mass (e.g. bone geometrical properties) tended to be larger in male compared to female bears. There were no differences (p>0.226) in bone structure, mineral content, or mechanical properties between fall and spring bears. Bone geometrical properties differed by less than 5% and bone mechanical properties differed by less than 10% between fall and spring bears. Porosity (fall: 5.5±2.2%; spring: 4.8±1.6%) and ash fraction (fall: 0.694±0.011; spring: 0.696±0.010) also showed no change (p>0.304) between seasons. Statistical power was high (>72%) for these analyses. Furthermore, bone geometrical properties and ash fraction (a measure of mineral content) increased with age and porosity decreased with age. These results support the idea that bears possess a biological mechanism to prevent disuse and age-related osteoporoses. 相似文献
993.
994.
Roosild TP Castronovo S Villoso A Ziemba A Pizzorno G 《Journal of structural biology》2011,176(2):229-237
Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer’s and Parkinson’s, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses. 相似文献
995.
Cloned ferrets produced by somatic cell nuclear transfer 总被引:10,自引:0,他引:10
Li Z Sun X Chen J Liu X Wisely SM Zhou Q Renard JP Leno GH Engelhardt JF 《Developmental biology》2006,293(2):439-448
Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. 相似文献
996.
Stasiukynas Diana C. Boron Valeria Hoogesteijn Rafael Barragán Jorge Martin Abigail Tortato Fernando Rincón Samantha Payán Esteban 《Acta ethologica》2022,25(3):179-183
acta ethologica - Common across various taxa, infanticide is a highly variable phenomenon present from insects to birds to mammals. In felids, antagonistic sexual coevolution led to the development... 相似文献
997.
998.
Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure 总被引:1,自引:2,他引:1 下载免费PDF全文
Ju-Ling Lin Samantha B. Joye Johannes C. M. Scholten Hendrik Sch?fer Ian R. McDonald J. Colin Murrell 《Applied microbiology》2005,71(10):6458-6462
Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition. 相似文献
999.
1000.
Daniel Crosby Melissa R. Mikolaj Sarah B. Nyenhuis Samantha Bryce Jenny E. Hinshaw Tina H. Lee 《The Journal of cell biology》2022,221(2)
ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion. 相似文献