Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation-2

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation"</p><p>http://www.biomedcentral.com/1471-2121/8/32</p><p>BMC Cell Biology 2007;8():32-32.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1952058.</p><p></p>of Ubc RNAi. Yellow color in the Merge columns indicates colocalization with aggregates. In control worms, ubiquitin and proteasomes colocalize to polyglutamine aggregates (top row). RNAi with eliminates localization of both ubiquitin and proteasomes. RNAi with does not disrupt localization of ubiquitin to aggregates, but does remove proteasomes, as expected. RNAi with either , or diminishes localization of ubiquitin and proteasomes to aggregates, whereas and show no effect on colocalization

Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation-1

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation"</p><p>http://www.biomedcentral.com/1471-2121/8/32</p><p>BMC Cell Biology 2007;8():32-32.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1952058.</p><p></p>achieve their normal size. Other research has indicated that aggregates initiate as small particles of aggregating protein that then travel along microtubules to ultimately collect and form large aggregates [42]. Since the knockdown of results in smaller, more numerous aggregates, ubiquitination by may be needed for small aggregates to come together and form large aggregates. UBC-2 and UBC-22 may normally have an inhibitory role in this process because their knockdown results in larger and less numerous aggregates. Ubiquitination by and may be required to recruit other proteins, such as proteasomes, to the aggregates, thereby allowing aggregates to further increase in size

Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation-5

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation"</p><p>http://www.biomedcentral.com/1471-2121/8/32</p><p>BMC Cell Biology 2007;8():32-32.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1952058.</p><p></p>lls. Synchronized worms from the Q82:GFP strain were treated with RNAi for 48 hours until they reached approximately the L4 larval stage. RNAi treatments included the knockdown of ubiquitin (), a subunit of the proteasome () and 11 different ubiquitin conjugating enzymes. Worms continue to express aggregates after these RNAi treatments, however, significant alterations in numbers and sizes of aggregates occur upon knockdown of specific Ubc's. A) Fluorescent images of control and RNAi treated worms showing the Q82:GFP aggregates. Scale bar is 100 μm. B) The average number of aggregates was counted manually under 40× magnification. RNAi knockdown of ubiquitin (), proteasome () or resulted in an increase in the number of aggregates whereas RNAi of either or showed a significant decrease in the number of aggregates. (* indicates statistical significance at p < .05). C) The average size of aggregates was determined following knockdown of each Ubc after 48 hours of RNAi treatment. The control was found to have an average aggregate area of 2.23 μm. Knockdown of , or resulted in a significant decrease in aggregate sizes. Knockdown of , or resulted in a significant increase in aggregate sizes. (* indicates statistical significance at p < .05). D) Same procedure as in A except that bacterial feeding cultures were combined to achieve knockdown of more than one Ubc. The phenotype is epistatic to both and . There is no additive effect between and . (* indicates statistical significance at p < .05). E) RT-PCR of RNAi treated worms confirms RNAi knockdown. Primers for , and were used to test RNA levels after RNAi treatments. For each set of primers, PCR template for lane is cDNA from control (pL4440) worms, lane ), lane ), and lane )

Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation-0

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation"</p><p>http://www.biomedcentral.com/1471-2121/8/32</p><p>BMC Cell Biology 2007;8():32-32.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1952058.</p><p></p>achieve their normal size. Other research has indicated that aggregates initiate as small particles of aggregating protein that then travel along microtubules to ultimately collect and form large aggregates [42]. Since the knockdown of results in smaller, more numerous aggregates, ubiquitination by may be needed for small aggregates to come together and form large aggregates. UBC-2 and UBC-22 may normally have an inhibitory role in this process because their knockdown results in larger and less numerous aggregates. Ubiquitination by and may be required to recruit other proteins, such as proteasomes, to the aggregates, thereby allowing aggregates to further increase in size

Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation-3

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation"</p><p>http://www.biomedcentral.com/1471-2121/8/32</p><p>BMC Cell Biology 2007;8():32-32.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1952058.</p><p></p>hort hairpin RNA plasmids (shRNA). HEK293 cells were transfected with Q81:YFP plasmid plus either control or Ubc shRNA plasmids. Two different shRNA constructs were used for each Ubc (designated as and in panels B-D). Cells were allowed to grow for 72 hrs after transfection. A) Following incubation, RNAi silenced cells were imaged by fluorescence microscopy. The pictures shown are an overlay between a bright field and a fluorescent image. B) Western blotting was performed to detect the level of knockdown of each Ubc using the antibodies indicated. All shRNA constructs achieved significant knockdown of their cognate Ubc. C) Aggregate size was measured after Ubc RNAi. Ube2A knockdown leads to smaller Q81:YFP aggregates however, UbcH5b and E2-25K knockdown increase the size of aggregates. At least 150 aggregates were measured for each shRNA transfection. These data represent the average sizes of aggregates from three independent experiments (> 450 aggregates). D) The number of aggregates per cell after Ubc RNAi was determined by analyzing photomicrographs of transfected cells. In control cells, approximately 42% of cells contain only soluble GFP and no aggregates. Ube2A knockdown causes a higher percentage of cells to form aggregates and also results in an increase in the percentage of cells with more than one aggregate. Knockdown of UbcH5b or E2-25K does not alter the ratio of cells containing aggregates versus cells with soluble GFP. Nor do they significantly affect the average number of cells per aggregate. Data were obtained from three independent experiments, counting 150 cells each time