Upon exposure to stress conditions, unfolded cell-surface nutrient transporters are rapidly internalized and degraded via the multivesicular body (MVB) pathway. Similarly, high concentrations of nutrients result in the downregulation of the corresponding transporters. Our studies using the yeast transporter Fur4 revealed that substrate-induced downregulation and quality control utilize a common mechanism. This mechanism is based on a conformation-sensing domain, termed LID (Loop Interaction Domain), that regulates site-specific ubiquitination (also known as degron). Conformational alterations in the transporter induced by unfolding or substrate binding are transmitted to the LID, rendering the degron accessible for ubiquitination by Rsp5. As a consequence, the transporter is rapidly degraded. We propose that the LID-degron system is a conserved, chaperone-independent mechanism responsible for conformation-induced downregulation of many cell-surface transporters under physiological and pathological conditions.
The multivesicular body (MVB) pathway delivers membrane proteins to the lumen of the vacuole/lysosome for degradation. The resulting amino acids are transported to the cytoplasm for reuse in protein synthesis. Our study shows that this amino acid recycling system plays an essential role in the adaptation of cells to starvation. Cells respond to amino acid starvation by up-regulating both endocytosis and the MVB pathway, thereby providing amino acids through increased protein turnover. Our data suggest that increased Rsp5-dependent ubiquitination of membrane proteins and a drop in Ist1 levels, a negative regulator of ESCRT activity, cause this response. Furthermore, we found that TORC1 and a second, unknown nutrient-sensing system are responsible for the starvation-induced protein turnover. Together, the data indicate that protein synthesis and turnover are linked by a common regulatory system that ensures adaptation and survival under nutrient-stress conditions.
Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.
ABS Materials has developed a new type of swellable organophilic material that extracts a wide array of dissolved hydrocarbons from oil field waters. With funding from the U.S. Department of Energy (SBIR Program) orchestrated through the National Energy Technology Laboratory, ABS Materials has been engineering systems to use swellable glass to treat flow back waters to remove dissolved organics. Two pilot scale systems were built: a non-regenerating skid-mounted system which handles inputs of up to 4 gal/min and a 60 gal/min trailer mounted system which included a mechanism for swellable glass regeneration.
Purpose To safely perform single‐iso multi‐target (SIMT) stereotactic radiosurgery (SRS), clinics must demonstrate SRS delivery accuracy for off‐axis targets. The traditional Winston–Lutz (W–L) was widely adopted because it provides a simple and accurate solution for testing radiation‐isocenter coincidence that uses a static target, enables testing arbitrary treatment angles, and does not require expensive commercial phantoms. The current noncommercial tests are cumbersome and insufficiently accurate. For an off‐axis Winston–Lutz (OAWL) test, one must design MLC fields centered on off‐axis targets. Unfortunately, because MLC leaf‐interfaces are often misaligned with the target center, accomplishing this presents a nontrivial geometry problem that has not been previously solved in the literature. We present a solution for evaluating SIMT SRS accuracy that provides a straightforward method for creating OAWL test fields and offers all the benefits of the standard W–L test. Methods We have developed a method to use any gantry, table, and initial collimator angles to create OAWL fields. This method calculates a series of nested coordinate transformations that produce a small collimator angle adjustment to align the MLC and create a symmetric field around an off‐axis target. Results For an 8 cm off‐axis target, the described method yields OAWL results within 0.07 mm of standard isocentric W–L results. Our six most recent isocentric W–L tests show max and mean errors of 0.59 and 0.37 mm, respectively. For six runs of our proposed OAWL test, the average max and mean errors are 0.66 and 0.40 mm, respectively. Conclusion This method accurately evaluates SRS delivery accuracy for off‐axis distances that span the majority of a typical human brain for a centered SIMT arc. We have made this method publicly available, so that physicists can employ it within their clinics, foregoing the need for expensive phantoms and improving access to the state‐of‐the‐art SIMT SRS technique.
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