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1.
Phys Rev Lett ; 132(24): 246701, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38949359

RESUMO

Recent experimental confirmation of spin inertia in ferromagnets positions this well-developed material class as a prime candidate for THz frequency applications. Spin-torque driven critical spin dynamics, such as auto-oscillations, play the central role in many spin-based technologies. Yet, the pressing question on spin inertia's effect on spin-torque driven dynamics in ferromagnets has remained unexplored. Here, we develop the theoretical framework of precessional auto-oscillations for ferromagnets with spin inertia. We discover and introduce the concept of nutational auto-oscillations and demonstrate that they can become pivotal for future ultrahigh frequency technologies. We conclude by revealing parallels between spin dynamics in ferrimagnets and inertial ferromagnets and derive an isomorphism that establishes a foundation for synergistic knowledge transfer between these research fields.

2.
Org Biomol Chem ; 20(3): 596-605, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-34951618

RESUMO

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.


Assuntos
Complexos de Coordenação/metabolismo , Európio/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Sulfotransferases/metabolismo , Ânions/química , Ânions/metabolismo , Cátions/química , Cátions/metabolismo , Complexos de Coordenação/química , Európio/química , Estrutura Molecular , Fosfoadenosina Fosfossulfato/química , Sulfotransferases/química , Fatores de Tempo
3.
Biochem J ; 477(6): 1159-1178, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32065231

RESUMO

Overexpression of S100P promotes breast cancer metastasis in animals and elevated levels in primary breast cancers are associated with poor patient outcomes. S100P can differentially interact with nonmuscle myosin (NM) isoforms (IIA > IIC > IIB) leading to the redistribution of actomyosin filaments to enhance cell migration. Using COS-7 cells which do not naturally express NMIIA, S100P is now shown to interact directly with α,ß-tubulin in vitro and in vivo with an equilibrium Kd of 2-3 × 10-7 M. The overexpressed S100P is located mainly in nuclei and microtubule organising centres (MTOC) and it significantly reduces their number, slows down tubulin polymerisation and enhances cell migration in S100P-induced COS-7 or HeLa cells. It fails, however, to significantly reduce cell adhesion, in contrast with NMIIA-containing S100P-inducible HeLa cells. When taxol is used to stabilise MTs or colchicine to dissociate MTs, S100P's stimulation of migration is abolished. Affinity-chromatography of tryptic digests of α and ß-tubulin on S100P-bound beads identifies multiple S100P-binding sites consistent with S100P binding to all four half molecules in gel-overlay assays. When screened by NMR and ITC for interacting with S100P, four chemically synthesised peptides show interactions with low micromolar dissociation constants. The two highest affinity peptides significantly inhibit binding of S100P to α,ß-tubulin and, when tagged for cellular entry, also inhibit S100P-induced reduction in tubulin polymerisation and S100P-enhancement of COS-7 or HeLa cell migration. A third peptide incapable of interacting with S100P also fails in this respect. Thus S100P can interact directly with two different cytoskeletal filaments to independently enhance cell migration, the most important step in the metastatic cascade.


Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Neoplasias/biossíntese , Tubulina (Proteína)/biossíntese , Animais , Células COS , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
4.
Physiol Rev ; 93(3): 1289-315, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23899565

RESUMO

Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.


Assuntos
Endopeptidases/classificação , Endopeptidases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Ubiquitina/metabolismo , Animais , Endopeptidases/química , Endopeptidases/genética , Modelos Moleculares , Conformação Proteica , Ubiquitina/química , Ubiquitinação/fisiologia
5.
Biochem J ; 475(15): 2417-2433, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-29934491

RESUMO

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.


Assuntos
Proteínas Aviárias/química , Heparitina Sulfato/química , Oligossacarídeos/química , Inibidores de Proteínas Quinases/química , Sulfotransferases/química , Quinases raf/antagonistas & inibidores , Animais , Proteínas Aviárias/genética , Galinhas , Heparitina Sulfato/farmacologia , Humanos , Oligossacarídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sulfotransferases/genética , Suínos , Quinases raf/química
6.
Nano Lett ; 17(1): 572-577, 2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28002674

RESUMO

Manipulation of magnetization by electric field is a central goal of spintronics because it enables energy-efficient operation of spin-based devices. Spin wave devices are promising candidates for low-power information processing, but a method for energy-efficient excitation of short-wavelength spin waves has been lacking. Here we show that spin waves in nanoscale magnetic tunnel junctions can be generated via parametric resonance induced by electric field. Parametric excitation of magnetization is a versatile method of short-wavelength spin wave generation, and thus, our results pave the way toward energy-efficient nanomagnonic devices.

7.
Biochim Biophys Acta ; 1842(11): 2049-59, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25035294

RESUMO

OBJECTIVES: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. METHODS: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. RESULTS: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3(+) Treg cells, while the number of RORγt(+) and (pTyr705)STAT3(+) cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. CONCLUSION: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

8.
J Biol Chem ; 288(37): 26697-708, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23897824

RESUMO

Shank/ProSAP proteins are major scaffold proteins of the postsynaptic density; mutations in the human SHANK3 gene are associated with intellectual disability or autism spectrum disorders. We have analyzed the functional relevance of several SHANK3 missense mutations affecting the N-terminal portion of the protein by expression of wild-type and mutant Shank3 in cultured neurons and by binding assays in heterologous cells. Postsynaptic targeting of recombinant Shank3 was unaltered. In electrophysiological experiments, both wild-type and L68P mutant forms of Shank3 were equally effective in restoring synaptic function after knockdown of endogenous Shank3. We observed that several mutations affected binding to interaction partners of the Shank3 ankyrin repeat region. One of these mutations, L68P, improved binding to both ligands. Leu-68 is located N-terminal to the ankyrin repeats, in a highly conserved region that we identify here as a novel domain termed the Shank/ProSAP N-terminal (SPN) domain. We show that the SPN domain interacts with the ankyrin repeats in an intramolecular manner, thereby restricting access of either Sharpin or α-fodrin. The L68P mutation disrupts this blockade, thus exposing the Shank3 ankyrin repeat region to its ligands. Our data identify a new type of regulation of Shank proteins and suggest that mutations in the SHANK3 gene do not necessarily induce a loss of function, but may represent a gain of function with respect to specific interaction partners.


Assuntos
Repetição de Anquirina/genética , Transtorno Autístico/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Animais , Transtorno Autístico/metabolismo , Proteínas de Transporte/metabolismo , Eletrofisiologia , Células HEK293 , Hipocampo/citologia , Humanos , Leucina/química , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Mutação de Sentido Incorreto , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Transmissão Sináptica , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinas/metabolismo
9.
J Biol Chem ; 288(12): 8238-8249, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23389036

RESUMO

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1-R13) organized into a compact cluster of four-helix bundles (R2-R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Adesões Focais/metabolismo , Proteínas de Membrana/química , Talina/química , Vinculina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Células Endoteliais da Veia Umbilical Humana , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Talina/metabolismo , Vinculina/metabolismo
10.
EMBO J ; 29(6): 1069-80, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20150896

RESUMO

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.


Assuntos
Integrinas/metabolismo , Talina/química , Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular , Integrinas/química , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Talina/metabolismo , Ubiquitina/metabolismo
11.
Mol Neurobiol ; 61(2): 693-706, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37656313

RESUMO

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.


Assuntos
Proteínas do Tecido Nervoso , Transdução de Sinais , Camundongos , Humanos , Animais , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo
12.
Essays Biochem ; 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38712401

RESUMO

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

13.
iScience ; 27(7): 110203, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39021807

RESUMO

Deleted in liver cancer 3 (DLC3) is a Rho GTPase-activating protein (RhoGAP) that plays a crucial role in maintaining adherens junction integrity and coordinating polarized vesicle transport by modulating Rho activity at the plasma membrane and endomembranes. By employing bioinformatical sequence analysis, in vitro experiments, and in cellulo assays we here identified a polybasic region (PBR) in DLC3 that facilitates the association of the protein with cellular membranes. Within the PBR, we mapped two serines whose phosphorylation can alter the electrostatic character of the region. Consequently, phosphomimetic mutations of these sites impaired the membrane association of DLC3. Furthermore, we found a new PBR-dependent localization of DLC3 at the midbody region, where the protein locally controlled Rho activity. Here, the phosphorylation-dependent regulation of DLC3 appeared to be required for proper cytokinesis. Our work thus provides a novel mechanism for spatiotemporal termination of Rho signaling by the RhoGAP protein DLC3.

14.
Sci Adv ; 10(34): eadi6286, 2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39167642

RESUMO

Tissue mechanical properties are determined mainly by the extracellular matrix (ECM) and actively maintained by resident cells. Despite its broad importance to biology and medicine, tissue mechanical homeostasis remains poorly understood. To explore cell-mediated control of tissue stiffness, we developed mutations in the mechanosensitive protein talin 1 to alter cellular sensing of ECM. Mutation of a mechanosensitive site between talin 1 rod-domain helix bundles R1 and R2 increased cell spreading and tension exertion on compliant substrates. These mutations promote binding of the ARP2/3 complex subunit ARPC5L, which mediates the change in substrate stiffness sensing. Ascending aortas from mice bearing these mutations showed less fibrillar collagen, reduced axial stiffness, and lower rupture pressure. Together, these results demonstrate that cellular stiffness sensing contributes to ECM mechanics, directly supporting the mechanical homeostasis hypothesis and identifying a mechanosensitive interaction within talin that contributes to this mechanism.


Assuntos
Matriz Extracelular , Homeostase , Talina , Talina/metabolismo , Talina/genética , Animais , Camundongos , Matriz Extracelular/metabolismo , Humanos , Mecanotransdução Celular , Mutação , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Aorta/metabolismo , Ligação Proteica , Fenômenos Biomecânicos
15.
bioRxiv ; 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38328095

RESUMO

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

16.
Nat Commun ; 15(1): 8002, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39266533

RESUMO

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.


Assuntos
Sistema de Sinalização das MAP Quinases , Mutação , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas p21(ras) , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Humanos , Camundongos , Linhagem Celular Tumoral , Feminino , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema de Sinalização das MAP Quinases/genética , Morte Celular/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Nus , Proteínas dos Microfilamentos
17.
J Phys Condens Matter ; 36(36)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38565125

RESUMO

Magnonicsis a research field that has gained an increasing interest in both the fundamental and applied sciences in recent years. This field aims to explore and functionalize collective spin excitations in magnetically ordered materials for modern information technologies, sensing applications and advanced computational schemes. Spin waves, also known as magnons, carry spin angular momenta that allow for the transmission, storage and processing of information without moving charges. In integrated circuits, magnons enable on-chip data processing at ultrahigh frequencies without the Joule heating, which currently limits clock frequencies in conventional data processors to a few GHz. Recent developments in the field indicate that functional magnonic building blocks for in-memory computation, neural networks and Ising machines are within reach. At the same time, the miniaturization of magnonic circuits advances continuously as the synergy of materials science, electrical engineering and nanotechnology allows for novel on-chip excitation and detection schemes. Such circuits can already enable magnon wavelengths of 50 nm at microwave frequencies in a 5G frequency band. Research into non-charge-based technologies is urgently needed in view of the rapid growth of machine learning and artificial intelligence applications, which consume substantial energy when implemented on conventional data processing units. In its first part, the 2024 Magnonics Roadmap provides an update on the recent developments and achievements in the field of nano-magnonics while defining its future avenues and challenges. In its second part, the Roadmap addresses the rapidly growing research endeavors on hybrid structures and magnonics-enabled quantum engineering. We anticipate that these directions will continue to attract researchers to the field and, in addition to showcasing intriguing science, will enable unprecedented functionalities that enhance the efficiency of alternative information technologies and computational schemes.

18.
J Struct Biol ; 184(1): 21-32, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23726984

RESUMO

Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods form a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.


Assuntos
Talina/química , Talina/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Citoesqueleto/química , Citoesqueleto/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
19.
J Biol Chem ; 287(10): 6979-90, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22235127

RESUMO

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin ß subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin ß tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin ß subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in αIIbß3 integrin activation, but not for kindlin binding to integrin ß tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well ß-integrin tails contribute to the ability of kindlins to activate integrins.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Proteínas de Transporte/genética , Adesão Celular/fisiologia , Cricetinae , Cricetulus , Proteínas do Citoesqueleto/genética , Adesões Focais/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Musculares/genética , Proteínas de Neoplasias/genética , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Estrutura Terciária de Proteína , Talina/genética , Talina/metabolismo
20.
Biochim Biophys Acta ; 1808(4): 1021-31, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21130070

RESUMO

Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.


Assuntos
Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Calorimetria , Membrana Celular/química , Membrana Celular/metabolismo , Dicroísmo Circular , Humanos , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosfoproteínas/química , Fosforilação , Potássio/metabolismo , Ligação Proteica , Serina/metabolismo , Sódio/metabolismo
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