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Individual globular domains and domain unfolding visualized in overstretched titin molecules with atomic force microscopy.
Full text linkTitin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin's globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme's ATP-binding site and hence contributing to the molecule's mechanosensory function.Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin's globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme's ATP-binding site and hence contributing to the molecule's mechanosensory function
Structural investigation of the molecular mechanisms underlying titin elasticity and signaling
Full text linkTitin is a giant protein that spans >1µm from the Z-disc to the M-line, forming an intrasarcomeric filament system in vertebrate striated muscle, which is not only essential for the assembly of the sarcomere, but also critical for myofibril signaling and metabolism. Furthermore, it provides the sarcomere with resting tension, elasticity and restoring forces upon stretch, ensuring the correct positioning of the actin-myosin motors during muscle function. Titin is composed of ~300 immunoglobulin (Ig) and fibronectin-III (FnIII) domains, arranged in linear tandems. They are interspersed by an auto-inhibited Ser kinase (TK) close to its C-terminus as well as several unique sequences, most prominently a differentially spliced stretch rich in PEVK residues which localizes to the I-band part of titin where its elastic properties reside. There, the PEVK segment is flanked by a long Ig tandem, which together act as serial molecular springs that determine titin elastic response.
The focus of this work lay in the elucidation of the molecular mechanisms governing titin I-band elasticity and the recruitment of the M-line signalosome around TK involved in the control of myofibril turnover and the trophic state of muscle. To that effect, we have elucidated the crystal structure of a six-Ig fragment representative of the elastic Ig-tandem at 3.3Å resolution. The model reveals the molecular principles of Ig-arraying at the skeletal I-band of titin as mediated by conserved Ig-Ig transition motifs. Regular domain arrangements within this fragment point at the existence of a high-order in the fine structure of the filament, which is confirmed by EM data on a 19-mer poly-Ig segment. Our findings indicate a long-range, supra-order in the skeletal I-band of titin, where assembly of Ig domains into dynamical super-motifs is essential for the elastic function of the filament. We propose a novel model of spring mechanism for poly-Ig elasticity in titin based on a “carpenter ruler” model of skeletal I-band architecture. Furthermore, we have focused on the recruitment of the ubiquitin ligase MURF1 to the M-line signalosome through its specific interaction with titin domains A168 A170. MuRF1 contains several oligomerization motifs in succession, which indicates a possible need for tight regulation. We have therefore analyzed their influence on the oligomeric state of the protein. Our SEC-MALS data showed that the a-helical region of MuRF1 is dimeric in isolation, while in combination with the preceding B-Box domain, itself a dimerization motif, higher-order assembly is induced, which might be of physiological importance. We could also show that higher-order assembly of MuRF1 did not disrupt binding to A168-A170 in pull-down assays. Further biophysical or structural characterization of the complex of A168-A170 with MuRF1 constructs was hindered by the severely compromised solubility of the complex. Finally, we have successfully solved the crystal structure of the FnIII-Kin-Ig region of twitchin, which corresponds to titin A170-TK-M1. The N-terminal linker wraps around the kinase domain and positions the preceding FnIII domain in such a way that it blocks the autoregulatory tail in its inhibitory positon. Thus, from the structure we could conclude that stretch-activation of Twc kinase seems unlikely and instead propose phosphorylation of Y 104 as a possible activation mechanism.
Our findings illustrate how the structural and functional diversity in titin’s modular architecture has evolved not only on the basis of individual domains. Rather, functionality often involves adaptation of several neighboring domains or even whole Ig tandems/super-repeats. This is reflected in variations in mechanical and dynamic properties observed in different parts of the chain and highlights the necessity of working with representative multi-domain fragments to gain a comprehensive understanding of the titin chai
Structural studies on protein scaffolds related to muscle physiology and disease : the titin filament, its associated component MuRF-1 and nuclear LAP2[alpha]
Full text linkThe titin molecule has a length of overµm and functions as a colossal protein scaffold in the muscle sarcomere. Up to 90% of its total mass is composed of repetitive immunoglobulin (Ig) and fibronectin (FnIII) domains that form linear tandems interspersed by unique sequences, among them a Ser/Thr kinase domain located at its C-terminus. The distinct pattern of Ig and FnIII motifs N-terminal to the kinase domain is conserved in other „giant kinases“ and invertebrate titin homologues. In vertebrate titin, it is involved in the specific recruitment of the ubiquitin ligase MuRF-1 to the filament. MuRF-1 is involved in the pathological atrophy of skeletal and cardiac muscle. We have determined the crystal structure of titin A168-A170 comprising two Ig and one FnIII domains and established its binding to MuRF-1 in solution. We analysed the structure with the aim to understand the interdomain relationships between repetitive Ig and FnIII subunits in titin as well as to shed light into the molecular determinants that confer specificity to ligand binding on the scaffold and in particular in the M-line interface to MuRF-1. A168-A170 shows an extended, rigid architecture. Its surface displays a shallow groove along its full length as well as a unique loop protrusion, both features conceivably mediating MuRF-1 binding. Moreover, our ITC data show that binding occurs with high affinity between residues 166-315 of MuRF-1. These data suggest that A168-A170 is of interest to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover. In addition we have elucidated the structure of the B-box domain of MuRF-1 to further investigate the role of MuRF-1 in homo- and hetero-oligomeric interactions at the M-line region. We found that MuRF-1 B-box adopts a RING-finger-like fold and exists in a dimeric state in solution. The domain possesses characteristic surface properties that are likely to mediate interactions of MuRF-1 with other sarcomeric components that are important in MuRF-1 function at the M-line. Finally, we have also carried out the biophysical characterization of the nuclear adaptor protein LAP2! that interacts with the nuclear lamina scaffold. Conceptually, LAP2! and the nuclear lamina are closely related systems to MuRF-1 and titin. This characterization, whose ultimate finality is to understand the interaction of LAP2! with lamin A/C establishes now the basis for a future structure elucidation.
This work illustrates how scaffold protein systems, which are structural skeletons
composed of multiple repetitive units, can become functionalized by the recruitment of
specific shuttle proteins to their surface. Specific binding in such systems involves
steric factors as well as the evolution of unique sequence inserts at defined locations.
Recruited proteins often act as adaptors that, in turn, attract other cellular components.
They often result in large, heterogeneous molecular assemblies that amplify the
physiological response. In the case of titin, the potential formation of a signalosome
assembly at its M-line, surrounding a kinase domain, is thought to mediate mechanotransduction
pathways involved in the regulation of myofibril turn-over and, thereby, in
the adaptative remodelling of muscle to mechanical load
Uji Aktivitas Antidiabetes Fraksi Air Kulit Buah Manggis (Garcinia mangostana L.) pada Mencit Putih Jantan Dengan Metode Induksi Aloksan; Titin Nur Farida, 062210101057
No full textDiabetes mellitus adalah suatu sindroma gangguan metabolisme dengan
hiperglikemia yang tidak semestinya sebagai akibat suatu defisiensi sekresi insulin
atau berkurangnya efektifitas biologis dari insulin atau keduanya. Dalam jangka
panjang, penyakit ini dapat mengakibatkan komplikasi, misalnya atherosclerosis
pada jantung, kaki dan otot, kerusakan saraf perifer, gangguan retina dan kerusakan
ginjal.
Salah satu pengobatan alternatif dalam mengobati penyakit diabetes mellitus
adalah dengan memanfaatkan bahan alam, seperti kulit buah manggis (Garcinia
mangostana L.) yang secara empiris digunakan oleh masyarakat untuk mengobati
diabetes mellitus. Untuk lebih memberikan dasar bagi bukti manfaatnya, perlu
dilakukan suatu penelitian.
Penelitian ini bertujuan untuk mengetahui aktivitas antidiabetes fraksi air kulit
buah manggis dengan berbagai dosis dan menentukan apakah terdapat perbedaan
aktivitas antidiabetes antar kelompok perlakuan. Prosedur pengujian aktivitas
antidiabetes fraksi air kulit buah manggis dalam penelitian ini adalah menggunakan
metode induksi aloksan. Hewan coba yang digunakan dalam penelitian ini adalah
mencit jantan Galur Balb-C. Aloksan adalah suatu senyawa yang sering digunakan
untuk penelitian diabetes menggunakan hewan coba. Hewan coba dikatakan diabetes
jika kadar glukosa darahnya lebih dari kadar glukosa normal pada mencit yaitu 62,8-
176 mg/dL. Bahan uji dikatakan memiliki aktivitas sebagai anti dibetes jika dapat
menurunkan kadar glukosa darah pada mencit diabetes. Pengukuran kadar glukosa
darah menggunakan alat GlucoDrTM blood glucose meter AGM-2200. Berdasarkan analisis menggunakan Anova Satu Arah dengan taraf
kepercayaan 95% dan dilanjutkan dengan uji Least Significant Different (LSD)
menunjukkan bahwa kelompok fraksi air kulit buah manggis dosis 125 mg/kgBB,
250 mg/kgBB, 500 mg/kgBB, dan 750 mg/kgBB memiliki aktivitas antidiabetes yang
sebanding dengan kontrol positif yaitu glibenklamid dengan dosis 1,3 mg/kgBB. Dari
hasil analisis menunjukkan adanya kecenderungan semakin tinggi dosis fraksi yang
diberikan maka semakin tinggi pula aktivitas antidiabetesnya. Perbedaan penurunan
kadar glukosa darah dari keempat dosis fraksi dikarenakan terdapatnya perbedaan
jumlah kandungan senyawa aktif yang berperan dalam aktivitas antidiabetes ataupun
dikarenakan faktor individu dari mencit yang menyebabkan hasil yang berbeda.
Perbandingan persen penurunan kadar glukosa darah antara dosis 125 mg/kgBB, 250
mg/kgBB, 500 mg/kgBB, dan 750 mg/kgBB masing-masing adalah 26,95%, 37,06%,
39,70%, dan 51,47%. Senyawa aktif yang diduga memiliki aktivitas sebagai
antidiabetes adalah flavonoid dan xanton. Karena kedua senyawa tersebut berfungsi
sebagai antioksidan maka kedua senyawa diduga mampu bekerja sama dalam
menurunkan kadar glukosa darah mencit. Tetapi untuk mengetahui batas
keamanannya, diperlukan penelitian yang lebih lanjut menggunakan uji toksisitas
Structural insights into the basis and evolution of interactions in multi-subunit protein assemblies. tryptophan synthase and titin FNIII-repeats
Full text linkCellular processes benefit from evolutionary shaping when optimized protein-protein interactions result in enhanced functionality. In fact, most cellular proteins are tightly embedded into biological networks that function following a modularity principle. Modularity, whether based on components as parts of stable protein complexes or as dynamic units that interact only transiently (as in signalling and metabolic cascades), facilitates the combinatorial generation of complexity in protein networks through the re-wiring of modules in addition to the diversification of individual proteins – thereby increasing the “evolvability” of the system. The mechanisms that drive the emergence and evolution of molecular recognition in protein networks remain unclear. It is difficult to justify such evolution on the basis of organismic advantage, since the latter might only be noticeable once full pathways and cascades have evolved. It is then likely that the evolution of protein-protein interactions is in the first instance driven by a molecular principle of local advantage to the protein system itself - for example, molecular stability. Unfortunately, it is difficult to gain insights into the evolution of protein-protein interactions since the pathways of evolutionary shaping normally let intermediates of evolution disappear. Subsequently, conclusions are more usually drawn from the comparison of proteins between different species and by mutagenesis probing. In the current study, we aim at gaining an insight into the evolutionary shaping of proteins surfaces for hetero-complex formation by studying two systems at an early stage of development: Tryptophan Synthase B2b (TrpB2b) from S. solfataricus and the modular interfaces of the poly-FNIII tandems in the muscle filament titin. In the case of TrpB2b, the evolution of inter-subunit communication is addressed in addition. Both structures have been elucidated using X-ray crystallography and a comparative analysis of their surfaces has been carried out. The architectural elements subjected to evolutionary pressure have been identified and conclusions on their relation to function and evolution have been drawn
Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation
No full textAims: Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants. Methods and results: We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants. Conclusion: Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation
Titin
No full textNucleophosmin (NPM1) interacts with other scaffolding proteins to bring together two or more proteins in a relatively stable configuration. This is done to maintain the liquid-like matrix of the nucleolus. It has been seen that titin has multiple structural elements that are similar in their characteristics to NPM1. Overall, we suspect that titin binds NPM1, in order to test, we will work to purify NPM1 and titin over the summer in order to test the potential binding domains of NMP1 within titin. This will be done by using surface plasmon resonance to screen the binding domains to NPM1. In addition, mass spectrometry is used to test the possible interaction between NPM1 and titin
Inferring the diameter of a biopolymer from its stretching response
Full text linkWe investigate the stretching response of a thick polymer model by means of extensive stochastic simulations. The computational results are synthesized in an analytic expression that characterizes how the force versus elongation curve depends on the polymer structural parameters: its thickness and granularity (spacing of the monomers). The expression is used to analyze experimental data for the stretching of various different types of biopolymers: polypeptides, polysaccharides, and nucleic acids. Besides recovering elastic parameters (such as the persistence length) that are consistent with those obtained from standard entropic models, the approach allows us to extract viable estimates for the polymers diameter and granularity. This shows that the basic structural polymer features have such a profound impact on the elastic behavior that they can be recovered with the sole input of stretching measurements
Modulation of Titin-Based Stiffness by Disulfide Bonding in the Cardiac Titin N2-B Unique Sequence
No full textAbstractThe giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titin's spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-Bus), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-Bus molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-Bus. We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart
Effect of titin phosphorylation on degradation of titin from skeletal muscles
No full textThe degradation of titin could make the myofibrillar fragmentation to improve meat tenderization during postmortem. This study aimed to investigate effect of phosphorylation on titin degradation. Protein kinase A (PKA) and alkaline phosphatase (AP) were added to crude titin extracted from ovine longissimus lumborum (LL) muscles. Phosphorylated/dephosphorylated titin were incubated with μ-calpain at 4 °C for 2 days. Results showed titin in AP group started degradation earlier than that in PKA and control groups. There were 20, 16 and 12 phosphorylated sites identified by iTRAQ in the PKA, control and AP group, respectively. 3D structure of dephosphorylated titin fragment was simulated and its molecular dynamics trajectory analysis was performed using Discovery StudioTM. The dihedral angle in AP group was less and the dephosphorylated fragment had a higher kinetic energy and total energy. We suggested that changes caused by AP treatment might make titin unstable, which easily degraded by μ-calpain
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