Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.     

Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca²⁺-loaded calmodulin (Ca²⁺/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca²⁺/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca²⁺/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with ¹⁵N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca²⁺-dependent manner with the Par17 N terminus. The reverse experiment with ¹⁵N-labeled Ca²⁺/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca²⁺/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK^796–815 complex. In vitro tubulin polymerization assays furthermore showed that Ca²⁺/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca²⁺/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca²⁺/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca²⁺ signaling with microtubule function.     

Genetic meta-analysis of 15,901 African Americans identifies variation in EXOC3L1 is associated with HDL concentration

Meta-analyses of European populations has successfully identified genetic variants in over 150 loci associated with lipid levels, but results from additional ethnicities remain limited. Previously, we reported two novel lipid loci identified in a sample of 7,657 African Americans using a gene-centric array including 50,000 SNPs in 2,100 candidate genes. Initial discovery and follow-up of signals with P < 10⁻⁵ in additional African American samples confirmed CD36 and ICAM1. Using an additional 8,244 African American female samples from the Women’s Health Initiative SNP Health Association Resource genome-wide association study dataset, we further examined the previous meta-analyses results by attempting to replicate 20 additional putative lipid signals with P < 10⁻⁴. Replication confirmed rs868213, located in a splice donor region of exocyst complex component 3-like 1 (EXOC3L1) as a novel signal for HDL (additive allelic effect β = 0.02; P = 1.4 × 10⁻⁸; meta-analyses of discovery and replication). EXOC3L1 is strongly expressed in vascular endothelium and forms part of the exocyst complex, a key facilitator of the trafficking of lipid receptors. Increasing sample sizes for genetic studies in nonEuropean populations will continue to improve our understanding of lipid metabolism.     

Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR^−/−) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR^−/− mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.     

Risks and advantages of using surface laser photogrammetry on free‐ranging marine organisms: a case study on white sharks Carcharodon carcharias

This study employed a non‐lethal measurement tool, which combined an existing photo‐identification technique with a surface, parallel laser photogrammetry technique, to accurately estimate the size of free‐ranging white sharks Carcharodon carcharias. Findings confirmed the hypothesis that surface laser photogrammetry is more accurate than crew‐based estimations that utilized a shark cage of known size as a reference tool. Furthermore, field implementation also revealed that the photographer's angle of reference and the shark's body curvature could greatly influence technique accuracy, exposing two limitations. The findings showed minor inconsistencies with previous studies that examined pre‐caudal to total length ratios of dead specimens. This study suggests that surface laser photogrammetry can successfully increase length estimation accuracy and illustrates the potential utility of this technique for growth and stock assessments on free‐ranging marine organisms, which will lead to an improvement of the adaptive management of the species.     

Temporal and spatial distribution of young Brachyplatystoma spp. (Siluriformes: Pimelodidae) along the rapids stretch of the Madeira River (Brazil) before the construction of two hydroelectric dams

Monthly (April 2009 to May 2010) bottom‐trawl sampling for Brachyplatystoma species along the rapids stretch of the Madeira River in Brazil revealed that Brachyplatystoma rousseauxii larvae and juveniles were present in low abundances in all areas and during all hydrological periods. The presence of larvae and juveniles throughout the hydrological cycle suggests asynchronous spawning in the headwaters of the Madeira River.