Nuclear structure and function are governed by lamins, which are intermediate filaments that mostly consist of α-helices. Different lamin assembly models have been proposed based on low resolution and fragmented structures. However, their assembly mechanisms are still poorly understood at the molecular level. Here, we present the crystal structure of a long human lamin fragment at 3.2 Å resolution that allows the visualization of the features of the full-length protein. The structure shows an anti-parallel arrangement of the two coiled-coil dimers, which is important for the assembly process. We further discover an interaction between the lamin dimers by using chemical cross-linking and mass spectrometry analysis. Based on these two interactions, we propose a molecular mechanism for lamin assembly that is in agreement with a recent model representing the native state and could explain pathological mutations. Our findings also provide the molecular basis for assembly mechanisms of other intermediate filaments.

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (mu'=0.058 h(-1)) than at pH 6.5 (mu'=0.028 h(-1)). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2'-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.

Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.

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