Fig 2: Proposed model of co-translational translocation events in the presence of CADA and DNAJC3. Top panel: The ERLEC1-P2A-mCD4 construct was translated by the ribosome and was targeted to the ER membrane with import into the ER lumen of the ERLEC1 protein (blue arrow) and insertion of mCD4 in the ER membrane (pink arrow). Under CADA pressure, ERLEC1 was prevented from being translocated (clogged translocon) while mCD4 could still get inserted into the ER membrane via its own SP, with even higher efficiency. When DNAJC3 was highly expressed, the ERLEC1 clogged translocon was rapidly cleared, allowing insertion of more mCD4 in the ER membrane while ERLEC1 translocation was consistently inhibited by CADA. Bottom panel: The ERLEC1-mCD4 construct was translated but the ER targeting and membrane insertion of mCD4 was at a very low level. Treatment with suboptimal levels of CADA stabilized the targeting complex and enhanced the targeting and membrane insertion of mCD4, whereas, at high CADA concentration, the translation and translocation of mCD4 were slightly down-regulated. The presence of high DNAJC3 levels rapidly removed (and degraded) the stalled ERLEC1 species (and downstream mCD4 protein) and prevented the stalled mCD4 from eventually getting inserted into the ER membrane, thus, intensifying the net down-modulating effect of CADA on its substrates.

Fig 3: NP releases P58IPK from its negative regulator Hsp40.A. Bacterially expressed and purified GST or GST-P58IPK fusion proteins were incubated with Glutathione Sepharose beads. The beads were washed and further incubated with the lysates from HEK293T cells, stably expressing either EGFP (lanes 1–3) or Myc-NP (lanes 4–6). The bound material eluted from washed beads was examined by western blot analysis using appropriate antibodies. Input represents the whole cell lysate. B. HEK293T cells were co-transfected with plasmids expressing FLAG-P58IPK along with either Myc-Hsp40 or Myc-NP. The cell lysates were immunoprecipitated using anti-Myc antibody. The western blot analysis of the whole cell lysate (WCL) (lanes 1–3) and immunoprecipiated material (lanes 4–6) was carried out using appropriate antibodies. C, D, E and F. Representative BLI sensograms showing over time association and dissociation of N protein with GST-P58IPK (C), N protein with GST tag (D), HSP40 with GST-P58IPK (E) and HSP40 with GST tag (F). The sensograms were generated at two concentrations of GST-P58IPK and GST tag, shown by red and blue color in panels C-F (see materials and methods for details). G. HEK293T cells were cotransfected with plasmids expressing FLAG-NP or Myc tagged wild type P58IPK or P58IPK mutants lacking either subdomain I composed of TPR repeats 1–3 (ΔTPR1-3) or both subdomains I and II composed of TPR repeats 1–6 (ΔTPR1-6). Cell lysates were Immunoprecipitated using anti-Myc antibody. Western blot analysis of the whole cell lysate (WCL) or immunoprecipitated material was carried out using either anti-FLAG or anti-Myc antibodies, as shown. H. The experiment shown in this panel was carried out exactly as in panel C except FLAG-NP was replaced with FLAG-Hsp40. I. Schematic representation of TPR domain of P58IPK. J. The kinetic profiles from panels C-F were analyzed for the calculation of binding parameters, as mentioned in Materials and Methods. Five percent of the calculated KD value was added as standard deviation.

Fig 4: Cell biological defects in the thyrocytes of TG+/cog mice.(A) Serum TSH and total T4 and T3 levels (n = 7–10 mice/group) and thyroid gland size (graph at right, area in mm2 normalized to body weight; n = 7–14 mice/group) of TG+/+, TG+/cog, and T4-treated TG+/cog mice. (B) Representative H&E images (upper images, n = 4–8 mice/group; bar = 40 μm) and anti-Tg immunofluorescence (lower images, n = 2–4 mice/group; bar = 50 μm) of thyroid glands from TG+/+, TG+/cog, and T4-treated TG+/cog mice. (C) mRNA levels of Tg (upper graph) and CHOP (lower graph) in the thyroid glands of TG+/+ and TG+/cog mice (normalized to 18S RNA; n = 5 mice/group). (D) Western blotting analysis of full-length (FL) and cleaved (CL) PARP (upper) and CHOP (lower) and quantitation (graph at right, CL PARP/FL PARP; CHOP protein normalized to actin) in the thyroid glands of TG+/+, TG+/cog, and T4-treated TG+/cog mice (n = 4–5 mice/group). (E) Western blotting analysis of BiP, ERdj6, phosphorylated eIF2α, total eIF2α, and Hrd1 (blots above, quantitation below, normalized to actin; except phosphorylated eIF2α normalized to total eIF2α) in the thyroid glands of TG+/+, TG+/cog, and T4-treated TG+/cog mice (n = 3–5 mice/group). (F) Representative TUNEL labeling (red) with DAPI counterstain (blue) and graph of TUNEL-positive follicles as a fraction of total thyroid follicles in thyroid sections of TG+/+, TG+/cog, and T4-treated TG+/cog mice (n = 5–7 mice/group). (G) Mock digest or endoglycosidase H (Endo H) digest followed by Tg Western blotting (gel at left) quantified (bar graph at right) from thyroid of TG+/+, TG+/cog, and T4-treated TG+/cog mice (n = 3–4 mice/group). All quantified data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA with Tukey’s post hoc test). Each dot represents an individual animal (squares = males; circles = females).

Fig 5: ER stress, cell death, and T4 synthesis in TGrdw/rdw rats.(A) Left: BiP, p58ipk, and phospho-eIF2α Western blotting in thyroids of WT (+/+) and TGrdw/rdw rats (each lane represent 1 animal). Right: Quantification (BiP and p58ipk normalized to tubulin; phospho-eIF2α normalized to total eIF2α; mean ± SD). **P < 0.01, ***P < 0.001 (unpaired 2-tailed Student’s t test). (B) CHOP mRNA levels (normalized to YWHAZ) in the thyroid glands of WT and TGrdw/rdw rats (n = 7–8 animals/group; each point represents 1 animal; mean ± SD). ***P < 0.001 (unpaired 2-tailed Student’s t test). (C) Top: Representative samples showing spliced and unspliced XBP1 mRNA in the thyroids of WT, TGrdw/+, and TGrdw/rdw rats (n = 3–6 animals/group; each lane represents 1 animal). Hprt1 was used as a loading control. Bottom: Quantitation of the fraction of spliced XBP1 (mean ± SD). **P < 0.01, ***P < 0.001 (1-way ANOVA, Bonferroni post hoc test). (D) Representative TUNEL staining and immunofluorescence of T4-containing protein with DAPI counterstain in the thyroids of WT and TGrdw/rdw rats (n = 4 animals/group). Scale bars: 20 μm. (E) Representative immunofluorescence of cleaved caspase-3 with DAPI counterstain in thyroids of WT and TGrdw/rdw rats (n = 5 animals/group). For clarity, a dashed white line delimits the thyroid follicle lumen in the WT rats (in which cleaved caspase-3 is not detectable). Scale bars: 20 μm. (F) Western blotting of PARP in thyroid glands from WT and TGrdw/rdw rats (n = 3–4; each lane represents 1 animal). (G) Left: Representative Western blotting of T4-containing protein in thyroid homogenates of WT and TGrdw/rdw rats (n = 5 animals/group) with or without soluble competitor T4 to block specific bands (left of dotted red line). Right: The same samples immunoblotted with mAb anti-Tg showing intentional overloading of the TGrdw/rdw rat sample.