179 research outputs found
Mechanically Induced Titin Kinase Activation Studied by Force-Probe Molecular Dynamics Simulations
No full textAbstractThe conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal β-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal β-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor
Cytoskeletal protein kinases: titin and its relations in mechanosensing
No full textTitin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation
Effects of motor deprivation on neurogenesis and muscle-derived neurotropic factors
No full textThe positive role of exercise in individuals affected by neurologic dis- eases was recently brought to light (Okonkwo et al. 2014). On the contrary, both astronauts and patients affected by movement-limiting pathologies face impairment in muscle and brain performance. More- over, recent evidences suggest that myokines released by exercising muscles affect the expression of brain derived neurotrophic factors (Phillips et al. 2014). The purpose of this work is to study how muscular inactivity affects neurogenesis and the factors that are involved in the interaction between the muscle and the neurogenic areas. We plan to study the effect of motor deprivation using a well-established ground based model: the hindlimb unloading model (HU). Four-month-old male C57BL/6 mice were randomly selected and assigned to control (CTRL) or HU groups. The unloading lasted 14 days. At the end of the suspen- sion, gastrocnemius (GAS), soleus (SOL) muscle and brain were dissected and processed for the appropriate experimental procedure. Various neurogenic regions of the mice central nervous system (CNS) were isolated and the evaluation of the proliferative capacity of neuronal stem cells (NSCs) was performed in cultures obtained from HU and CTRL mice. Signals involved in controlling metabolism were studied in both muscles and brain. Muscle expression of fibronectin type III domain-containing protein 5 (FNDC5), which seems to play a role in regulating brain-derived neurotrophic factor (BDNF) production in CNS (Wrann et al. 2013), was also assessed. Preliminary results indicate that HU NSCs have a reduced capacity to proliferate and differentiate compared to CTRL. A reduction of PGC1alfa and FNDC5 expression in HU SOL was found. The study reveals that motor deprivation impairs neurogenesis and muscle neurotrophic factors expression
The sarcomeric cytoskeleton: who picks up the strain?
No full textIn striated muscle sarcomeres, the contractile actin and myosin filaments are organised by a subset of specialised cytoskeletal proteins, the sarcomeric cytoskeleton. They include α-actinin, myomesin, and the giant proteins titin, obscurin and nebulin, which combine architectural, mechanical and signalling functions. Mechanics and signalling in the sarcomere appear tightly interdependent, but the exact contributions of the various sarcomeric cytoskeleton proteins to strain handling or signalling are only just emerging. General mechanisms of cytoskeletal mechanics and signalling may be gleaned from the sarcomere as a specialised actomyosin system. Recent work has led to insight into the interactions, structure, and mechanical stability of sarcomeric protein complexes that fulfil both structural and signalling roles.</p
Tropomodulins and tropomyosins: working as a team
No full textActin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tmod and TM are crucial constituents of the actin filament network, making their presence indispensable in living cells. Tropomyosin (TM) is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin (Tmod), which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tmod consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two TM-binding sites and one TM-dependent actin-binding site, whereas the C-terminal domain contains a TM-independent actin-binding site. Tmod binds to two TM molecules and at least one actin molecule during capping. The interaction of Tmod with TM is a key regulatory factor for actin filament organization. The binding efficacy of Tmod to TM is isoform-dependent. The affinities of Tmod/TM binding influence the proper localization and capping efficiency of Tmod at the pointed end of actin filaments in cells. Here we describe how a small difference in the sequence of the TM-binding sites of Tmod may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod–TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure
Single-Molecule Force Spectroscopy Reveals the Function of Titin Kinase as Force Sensor
No full textMechanoenzymatics of titin kinase
No full textBiological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATIP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnove
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