Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.
Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.
Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following is the most likely mechanism by which the sb region of Asb-4 decreases IRS4 levels?

Question

Passage
The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting. In addition, the protein insulin receptor substrate 4 (IRS4) , known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4. Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection. HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively) . Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away. The bound proteins were then visualized by Western blot using α-myc antibodies. The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb) . Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1.

Figure 1 Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub) . The results are shown in Figure 2.

Figure 2 Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control) , myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid
Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95.
-Which of the following is the most likely mechanism by which the sb region of Asb-4 decreases IRS4 levels?

Quizplus · Accepted Answer

11ecba2d_0eff_c320_a3bf_c57779b7f3d6_MD0008_00 Protein levels within a cell can be controlled by both the rate of protein synthesis (expression) and the rate of protein degradation. Protein expression is controlled by several mechanisms, including transcription factors and RNA processing. Degradation is typically induced by targeting proteins to the lysosome or the proteasome. Several pieces of evidence indicate the mechanism by which the sb domain of Asb-4 regulates IRS4 levels. First, Figure 1 shows that the level of flag-IRS4 decreases in proportion to the level of myc-Asb-4 expression. Therefore, Asb-4 causes a decrease in IRS4 levels, either by inhibiting expression or by inducing degradation. When the sb domain is removed (myc-Asb-4Δsb), IRS4 levels are constant, indicating that the sb domain of Asb-4 is required for regulation of IRS4. Second, Figure 2 shows that when myc-Asb-4 is present, a ubiquitin tag is detected. Because this experiment was performed by binding proteins to beads containing the α-flag antibody, all proteins other than flag-IRS4 were washed away or bound to beads indirectly, most likely by interacting with IRS4. Figure 2 shows that ubiquitin binds to IRS4 only when the sb domain is present. Therefore, it is likely that Asb-4 causes ubiquitination of flag-IRS4. Ubiquitination does not occur when the sb domain is removed, indicating that this domain is required for ubiquitination. Proteins with ubiquitin tags are typically targeted to the proteasome for destruction. Based on all the data together, the sb domain of Asb-4 most likely regulates IRS4 levels by targeting IRS4 to the proteasome for destruction. (Choices B and C) If the sb domain induced IRS4 expression or inhibited IRS4 proteolysis (degradation), IRS4 levels would be expected to increase, not decrease, when the sb domain is present. (Choice D) Ubiquitin tags target proteins to the proteasome, not the lysosome, for destruction. The lysosome typically degrades secretory proteins, whereas the passage states that IRS4 is cytosolic. Educational objective: Protein levels in a cell are controlled by expression and degradation rates. Expression is controlled by mechanisms such as transcription factors and RNA processing, whereas degradation is controlled by targeting secretory proteins to the lysosome and cytosolic proteins to the proteasome. Ubiquitin tags mark cytosolic proteins for proteasomal destruction.

Passage The Ankyrin Repeat and SOCS Box Containing Protein 4 (Asb-4)