Journal: bioRxiv

Article Title: Nascent Chain Ubiquitination is Uncoupled from Degradation to Enable Protein Maturation

doi: 10.1101/2023.10.09.561585

Figure Lengend Snippet: (A) A schematic diagram illustrates the ribosome stalling induced by XBP1u arrest peptide (AP). The incorporation of the last asparagine (N) residue is inhibited due to the stall. The mutation of L246 and W256 to alanine residues relieves the stall. (B) WT 293T cells or LTN1 KO (LKO) cells expressing empty vector or XBP1u were fractionated by 10-30% sucrose density gradient and the fractions were analyzed by immunoblotting for the indicated antigens. (C) Cells expressing empty vector or FLAG-tagged XBP1u were lysed and immunoprecipitated (IPed) for XBP1u using anti-FLAG beads and analyzed by immunoblotting. (D) WT and LKO cells were treated with MG132 (20μM) for the indicated time points and analyzed by immunoblotting. (E, F) Pulse-chase analysis of WT and LKO cells expressing XBP1u or XBP1u-AP*. The data were quantified from two independent experiments are represented as means ±SEMs. (G) Empty vector or FLAG-XBP1u is transfected along with HA-ubiquitin into WT or LKO cells were immunoprecipitated with anti-FLAG beads. The resulting samples were probed for ubiquitinated proteins (Ub-proteins) using an HA antibody and for XBP1u with an anti-FLAG antibody. (H) The indicated cells expressing FLAG-XBP1u-AP* were analyzed as in (G). (I) Cells expressing FLAG-XBP1u or FLAG-XBP1u-AP were analyzed as in (G), but IP samples were probed with an K48-linked ubiquitin chain specific antibody. See also Figure S1

Article Snippet: Antibodies used for immunoblotting are as follows: Rabbit α-LTN1 (Proteintech; #28452-1-AP); Rabbit α-NEMF (Bethyl; # A305-758A), rat α-FLAG L5 (BioLegend; #12775); mouse α-HA-HRP (Cell Signaling Technology; #2999S); mouse α-tubulin (Abcam; #ab7291); goat α-rat-HRP (Cell Signaling Technology; #7077), goat α-mouse-HRP (Jackson ImmunoResearch; #115-035-003); goat α-rabbit-HRP (Jackson ImmunoResearch; #111-035-003); goat α-rat IgG-Cy2 (Jackson ImmunoResearch; #112–225-167); Rabbit α-XBP1u (Cell signaling Technology; # #12782).

Techniques: Residue, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Pulse Chase, Transfection, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Nascent Chain Ubiquitination is Uncoupled from Degradation to Enable Protein Maturation

doi: 10.1101/2023.10.09.561585

Figure Lengend Snippet: (A) 293T cells expressing the indicated HA-tagged constructs were subjected to anti-HA magnetic beads and analyzed by immunoblotting for the indicated antigens. (B) FLAG-XBP1u or its mutant were transfected along with HA-ubiquitin into cells treated with either control siRNA or RNF126 siRNA. Cell lysates were IPed with anti-FLAG beads and probed for Ub-proteins using an HA antibody and for XBP1u with an anti-FLAG antibody. (C) CHX chase analysis was performed in cells treated with indicated siRNA and expressing XBP1u or XBP1u-AP*, followed by immunoblotting for the indicated antigens. (D) (Top) Model construct illustrating the destabilized domain (dd) comprising FKBP12 fused with the arrest peptide (AP) of XBP1u. (Bottom) Schematic representation of LTN1-mediated ubiquitination of ddFKBP12-AP nascent chain. Shield1 small molecule is known to stabilize the folding of ddFKBP12. (E) WT or LTN1 KO cells expressing dd FKBP12-AP alongside HA-ubiquitin were untreated or treated with Shield1, immunoprecipitated, and analyzed by immunoblotting. (F) Pulse-chase experiments were conducted using cells expressing ddFKBP12-AP, treated either with or without Shield1. See also Figure S5

Article Snippet: Antibodies used for immunoblotting are as follows: Rabbit α-LTN1 (Proteintech; #28452-1-AP); Rabbit α-NEMF (Bethyl; # A305-758A), rat α-FLAG L5 (BioLegend; #12775); mouse α-HA-HRP (Cell Signaling Technology; #2999S); mouse α-tubulin (Abcam; #ab7291); goat α-rat-HRP (Cell Signaling Technology; #7077), goat α-mouse-HRP (Jackson ImmunoResearch; #115-035-003); goat α-rabbit-HRP (Jackson ImmunoResearch; #111-035-003); goat α-rat IgG-Cy2 (Jackson ImmunoResearch; #112–225-167); Rabbit α-XBP1u (Cell signaling Technology; # #12782).

Techniques: Expressing, Construct, Magnetic Beads, Western Blot, Mutagenesis, Transfection, Ubiquitin Proteomics, Control, Immunoprecipitation, Pulse Chase

Journal: bioRxiv

Article Title: Nascent Chain Ubiquitination is Uncoupled from Degradation to Enable Protein Maturation

doi: 10.1101/2023.10.09.561585

Figure Lengend Snippet: Upon release from stalled ribosomes, nascent proteins polyubiquitinated by the LTN1 E3 ligase are promptly deubiquitinated in the cytosol. This decoupling from immediate proteasomal degradation provides a secondary chance for some nascent proteins to achieve proper folding and maturation. However, if a nascent protein fails to attain stability and exposes a degron, it undergoes re-ubiquitination through cytosolic quality control mechanisms, leading to proteasomal degradation. It is conceivable that the nascent protein may re-enter a similar cycle, but ultimately, it would face degradation by the proteasome if it remains exposed to a degron for an extended period.

Article Snippet: Antibodies used for immunoblotting are as follows: Rabbit α-LTN1 (Proteintech; #28452-1-AP); Rabbit α-NEMF (Bethyl; # A305-758A), rat α-FLAG L5 (BioLegend; #12775); mouse α-HA-HRP (Cell Signaling Technology; #2999S); mouse α-tubulin (Abcam; #ab7291); goat α-rat-HRP (Cell Signaling Technology; #7077), goat α-mouse-HRP (Jackson ImmunoResearch; #115-035-003); goat α-rabbit-HRP (Jackson ImmunoResearch; #111-035-003); goat α-rat IgG-Cy2 (Jackson ImmunoResearch; #112–225-167); Rabbit α-XBP1u (Cell signaling Technology; # #12782).

Techniques: Ubiquitin Proteomics, Control

Journal: Brain : a journal of neurology

Article Title: Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

doi: 10.1093/brain/awac479

Figure Lengend Snippet: Figure 1 Non-stop mRNA protein products are degraded by the RQC in a Listerin-dependent manner in mammalian cells. (A) Schematic of stalling reporters and EGFP constructs. (B) GFP versus RFP expression profile of stalling reporter with poly-A sequences compared to a no insert control (left). Ratio of median fluorescence intensity of RFP to GFP shows that poly-A sequences induce stalling independent of listerin. Data are shown as mean ± standard error of the mean (SEM) with all data points (two-way ANOVA, n = 3, reporter P < 0.0001, LTN1 KO P = 0.9771, interaction P = 0.8548) (C) Confocal images of HEK 293T and LTN1 KO cells expressing EGFP-NS reporter show accumulation and reduced clearance of NS protein in LTN1 KO cells over time (scale bar = 50 µm). (D) Listerin overexpression in LTN1 KO cells significantly reduces levels of NS reporter protein in cells quantified by flow cytometry. Percentage is shown as mean ± SEM (mixed effects model, n = 3, Ltn1:HEK 293T P = 0.1246, Ltn1:LTN1 KO #1 P = 0.0001, Ltn1:LTN1 KO #2 P < 0.0001).

Article Snippet: For listerin rescue experiments, the LTN1_pCSdest plasmid used was a gift from Roger Reeves (#53855, Addgene).52 Plasmids expressing VCP wildtype (WT) or dominant-negative mutant (D2) were previously described.53 For NEMF antibody validation experiments, a plasmid expressing C-terminally HA tagged NEMF synthesized by GenScript was used. pDsRed-monomer-N1 plasmid was used as a transfection control.

Techniques: Construct, Expressing, Control, Fluorescence, Over Expression, Flow Cytometry

Journal: Brain : a journal of neurology

Article Title: Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

doi: 10.1093/brain/awac479

Figure Lengend Snippet: Figure 5 NEMF is recruited to DPR inclusions. (A) Representative confocal images of colocalization of NEMF and R-rich DPR proteins (arrowheads) ex pressed in HEK 293T cells (scale bar = 10 µm). (B) Representative confocal images of colocalization of NEMF and R-rich EGFP-DPR reporters (arrowheads) in I3N neurons (scale bar = 5 µm). (C) Confocal images show colocalization of DPR inclusions (poly-GA and poly-GR) and NEMF in frontal cortex and cerebellar granular layer of C9orf72 human tissue (scale bar, 5 µm). (D) Representative double immunofluorescence confocal images of DPR inclusions (poly-GA and poly-GR) and RQC factors (listerin and VCP) in frontal cortex of C9orf72-mutation carriers (scale bar = 5 µm). (E) Quantification of propor tion of DPR inclusions (poly-GA and poly-GR) that co-localize with RQC complex factors NEMF, listerin and VCP in frontal cortex and cerebellum from five C9orf72-expansion cases (total number of inclusions counted for poly-GA n = 90–130 or n = 1049–1201 and for poly-GR n = 41–55 or n = 62–114 in front al cortex and cerebellum, respectively; frontal cortex: Fisher’s exact test, NEMF P < 0.0001, listerin P = 0.0668 and VCP P = 0.6507; cerebellum: Fisher’s exact test, NEMF P < 0.0001, listerin P = 0.0767 and VCP P = 0.1403).

Article Snippet: For listerin rescue experiments, the LTN1_pCSdest plasmid used was a gift from Roger Reeves (#53855, Addgene).52 Plasmids expressing VCP wildtype (WT) or dominant-negative mutant (D2) were previously described.53 For NEMF antibody validation experiments, a plasmid expressing C-terminally HA tagged NEMF synthesized by GenScript was used. pDsRed-monomer-N1 plasmid was used as a transfection control.

Techniques: Immunofluorescence, Mutagenesis