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Jari Ylänne

Publications and source records attributed to Jari Ylänne.

12 recordsLinked to original sources
PubMed2006

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations.

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.

Actinin↗
PubMed2006

The molecular basis of filamin binding to integrins and competition with talin.

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.

Amino Acid Sequence↗
PubMed2005

The structure of the GPIb-filamin A complex.

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.

Contractile Proteins↗
PubMed2005

Structural basis for vertebrate filamin dimerization.

Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.

Actins↗
PubMed2004

Expression, purification and preliminary crystallographic characterization of a novel segment from the neurofibromatosis type 1 protein.

Neurofibromin (MW 320 kDa) is the protein responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most common genetic diseases worldwide. The neurofibromin GAP-related domain (GRD, MW 38 kDa) possess a Ras-specific GTPase-activating protein property, which is at present its only clear biochemical function. This article describes the study of the bacterial production and preliminary X-ray crystallographic analysis of a neurofibromin fragment located at the C-terminal end of the GRD, which contains a region reported to be homologous to the yeast Sec14p lipid exchange protein. Of the three crystal variants obtained, a tetragonal form diffracted to a resolution of at least 2.3 A.

Animals↗
PubMed2004

The ZASP-like motif in actinin-associated LIM protein is required for interaction with the alpha-actinin rod and for targeting to the muscle Z-line.

The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. alpha-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with alpha-actinin. Actinin-associated LIM protein (ALP), C-terminal LIM domain protein (CLP36), and Z band alternatively spliced PDZ-containing protein (ZASP) have a conserved region named the ZASP-like motif (ZM) between PDZ and LIM domains. To study the interactions and function of ALP we used purified recombinant proteins in surface plasmon resonance measurements. We show that ALP and alpha-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the alpha-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of alpha-actinin. This is the first indication that the ZM motif would have a direct role in a protein-protein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of alpha-actinin 2 that would strengthen the Z-line integrity.

Actinin↗
PubMed2004

Distinct roles of ligand affinity and cytoskeletal anchorage in alphaIIbbeta3 (GP IIb/IIIa)-mediated cell aggregation and adhesion.

Platelet integrin alphaIIbbeta3 (GP IIb/IIla) is functionally modulated by changes in ligand affinity or in cytoskeletal anchorage. CHO cells transfected with wild-type/mutated alphaIIbbeta3 allow the dissection of the relative contributions of the two regulatory mechanisms in alphaIIbbeta3-mediated adhesion and aggregation. Mutations included a truncation of the cytoplasmic domain of the beta-subunit, resulting in a loss of cytoskeletal anchorage of alphaIIbbeta3, and a VGFFK-deletion of the alpha-subunit, resulting in a permanent high affinity state. alphaIIbbeta3-mediated cell aggregation is dependent on the high affinity state but only partially on the cytoskeletal anchorage of alphaIIbbeta3. In contrast, alphaIIbbeta3-mediated cell adhesion is dependent on the cytoskeletal anchorage but only partially on the high affinity state of alphaIIbbeta3. Thus, the functional evaluation of mutated alphaIIbbeta3 implies a differential role of affinity state and cytoskeletal anchorage for alphaIIbbeta3-mediated cell adhesion and aggregation.

Animals↗
PubMed2003

Actin binding of a minispectrin.

A "minispectrin" has been constructed from the tail end of the alpha/beta heterodimer, and its actin-binding properties have been characterised. It is a complex of the N-terminal fragment of the beta-subunit consisting of the actin-binding domain plus the two first triple-helical repeats beta 1 and beta 2, and the C-terminal fragment of the alpha-subunit containing the repeats alpha 19 and alpha 20 plus the calmodulin-like domain. This minispectrin exists in a dimeric form that contains one copy of each polypeptide and binds to actin in a cooperative manner with an apparent K(d) of 2.5 microM. Calcium seems not to have any effect on its binding to actin. Electron microscopic analysis shows that the minispectrin decorates actin filaments as clusters, and induces formation of actin bundles. This study shows that the actin-binding region of the spectrin alpha/beta heterodimer retains its functional properties in a truncated form and establishes basis for further research on spectrin's structure and function.

Actinin↗
PubMed2002

The spectrin repeat: a structural platform for cytoskeletal protein assemblies.

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.

Actinin↗
PubMed2002

Pathways and intermediates in forced unfolding of spectrin repeats.

Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

Animals↗
PubMedDate not supplied

Adhesion of monocytes to medical steel as used for vascular stents is mediated by the integrin receptor Mac-1 (CD11b/CD18; alphaM beta2) and can be inhibited by semiconductor coating.

Implantation of stents into stenosed arteries helps to restore normal blood flow in ischemic organs. However, limited biocompatibility of the applied medical steel can cause acute thrombosis and long-term restenosis. Adhesion of monocytes to stent metal may participate in those acute and long-term complications of stent placement. Based on described prominent electrochemical properties of the interaction between the monocyte integrin receptor Mac-1 and its various ligands, we hypothesized, that this receptor is a central mediator of monocyte adhesion to stent metal and that semiconductor coating of medical steel reduces monocyte adhesion. Adhesion of monocytes on L-316 stainless steel was directly evaluated by light microscopy. Mac-1 could be identified as mediator of monocyte adhesion, since cell adhesion could be blocked by anti-Mac-1-antibodies, including the cross-reacting anti-GPIIb/IIIa antibody fragment abciximab. To further prove the central role of Mac-1, two CHO cell lines were generated expressing recombinant Mac-1 either as wild type, resulting in a low affinity receptor, or mutant with a GFFKR deletion of the alpha(M) subunit, resulting in a high affinity receptor. Indeed, adhesion was specific for Mac-1 and dependent on the affinity state of this integrin. Finally, we could demonstrate that Mac-1-mediated adhesion of monocytes to stents can be significantly inhibited by silicon carbide coating of the stent metal. In conclusion, the integrin Mac-1 and its affinity state could be identified as major mediators of monocyte adhesion on medical steel. As therapeutic strategies, the blockade of Mac-1 by antibodies or silicon carbide coating of steel inhibits monocyte adhesion on stents.

Adhesiveness↗