Substantial evidence indicates that endothelial dysfunction plays a critical role in atherogenesis. We previously demonstrated that apolipoprotein(a) (apo(a); the distinguishing protein component of the atherothrombotic risk factor lipoprotein(a)) elicits rearrangement of the actin cytoskeleton in human umbilical vein endothelial cells, characterized by increased myosin light chain (MLC) phosphorylation via a Rho/ Rho kinase-dependent signaling pathway. Apo(a) contains kringle (K)IV and KV domains similar to those in plasminogen: apo(a) contains 10 types of plasminogen KIV-like sequences, followed by sequences homologous to the plasminogen KV and protease domains. Several of the apo(a) kringles contain lysinebinding sites (LBS) that have been proposed to contribute to the pathogenicity of Lp(a). Here we demonstrate that apo(a)-induced endothelial barrier dysfunction is mediated via a Rho/ Rho kinase-dependent signaling pathway that results in increased MYPT1 phosphorylation and hence decreased MLC phosphatase activity, thus leading to an increase in MLC phosphorylation, stress fiber formation, cell contraction, and permeability. In addition, studies using recombinant apo(a) variants indicated that these effects of apo(a) are dependent on sequences within the C-terminal half of the apo(a) molecule, specifically, the strong LBS in KIV 10 . In parallel experiments, the apo(a)-induced effects were completely abolished by treatment of the cells with the lysine analogue ⑀-aminocaproic acid and the Rho kinase inhibitor Y27632. Taken together, our findings indicate that the strong LBS in apo(a) KIV 10 mediates all of our observed effects of apo(a) on human umbilical vein endothelial cell barrier dysfunction. Studies are ongoing to further dissect the molecular basis of these findings.Studies have shown that elevated concentrations of plasma lipoprotein(a) (Lp(a)) 2 (Ͼ30 mg/dl or Ͼ100 nM) are a risk factor for a variety of vascular diseases, including coronary heart disease, ischemic stroke, and venous thrombosis (1, 2). Lp(a) is identical to low density lipoprotein (LDL) in both lipid composition as well as the presence of apolipoproteinB-100. However, Lp(a) is clearly distinguishable from LDL by the presence of the unique glycoprotein apolipoprotein(a) (apo(a)) that is disulfide-linked to apolipoproteinB-100 in LDL by a single disulfide bond (3). Apo(a) bears a striking homology with plasminogen and contains multiple repeats of a sequence that resembles plasminogen kringle IV as well as sequences homologous to the kringle V and protease regions of plasminogen (4). The protease domain in apo(a) cannot be activated by activators of plasminogen; therefore, it cannot develop protease activity and hence lacks fibrinolytic activity (5). The kingle IV-like domain in apo(a) is classified into 10 types (KIV 1-10 ); the KIV 2 sequence is present in a variable number of identically repeated copies (from 3 to Ͼ40) giving rise to Lp(a) isoform size heterogeneity (6, 7). Kringle IV types 5-8 (KIV 5-8 ) possess "weak" lysinebinding s...

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.

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