Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or "conformational" phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural "LIR" motifs and selective cellular "cargo receptors" as plausible P450-ALD determinants

Durand et al 2012 Supplemental figures

Objective. Our objective was to compare the osteoclastogenic capacity of peripheral blood mononuclear cells (PBMCs) from patients with osteoarthritis (OA) to that of PBMCs from self-reported normal individuals. Methods. PBMCs from 140 patients with OA and 45 healthy donors were assayed for CD14+ expression and induced to differentiate into osteoclasts (OCs) over 3 weeks in vitro. We assessed the number of the OCs, their resorptive activity, OC apoptosis, and expression of the following cytokine receptors: receptor activator of nuclear factor κB (RANK), interleukin-1 receptor type I (IL-1R1) and IL-1R2. A ridge logistic regression classifier was developed to discriminate OA patients from controls. Results. PBMCs from OA patients gave rise to more OCs that resorbed more bone surface than did PBMCs from controls. The number of CD14+ precursors was comparable in both groups, but there was less apoptosis in OCs obtained from OA patients. Although no correlation was found between osteoclastogenic capacity and clinical or radiologic scores, levels of IL-1R1 were significantly lower in cultures from patients with OA compared to controls. OC apoptosis and expression levels of IL-1R1 and IL-1R2 were used to build a multivariate predictive model for OA. Conclusion. During 3 weeks of culture under identical conditions, monocytes from patients with OA display enhanced capacity to generate OCs compared to cells from controls. Enhanced osteoclastogenesis is accompanied by increased resorptive activity, reduced OC apoptosis and diminished IL-1R1 expression. These findings support the possibility that generalized changes in bone metabolism affecting OCs participate in the pathophysiology of OA

The evolutionarily conserved long non‐coding RNA <i>LINC00261</i> drives neuroendocrine prostate cancer proliferation and metastasis <i>via</i> distinct nuclear and cytoplasmic mechanisms

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+/PSA+) or NEPC (AR-/SYN+/CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease

Mobilization of Procollagen and Lysosomes during Osteoblast Stimulation with Ascorbic Acid

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).Ph

Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

<div><p>Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.</p> </div

qRT-PCR analysis of mRNA expression levels of three identified Rab GTPases in OBs treated with AA for 6 hours.

<p>Quantitative PCR analysis was performed on mRNA from control and 6 hour AA-stimulated MC3T3-E1 osteoblasts. The graph shows the signal intensity of OB Rab GTPase expression normalized against that of GAPDH from 3 independent experiments. Data is reported as mean ± SEM A significant increase in Rab1, 3d, and 27b mRNA expression levels after 6 hours of AA treatment versus no stimulation of control cells. * p<0.05.</p

Procollagen localizes to the ER and translocates to the Golgi after AA-stimulation of OBs.

<p>(A) Control and 6 hour AA differentiated MC3T3-E1 cells were fixed and stained with procollagen antibody shown in red and ER (PDI) antibody in green. (B) Same experiment as (A) except that the Golgi (GM130) was immunostained in green. Procollagen resides in the ER in undifferentiated control cells and upon addition of AA is translocated to the Golgi. (C) Treatment of cells with 0.5 µg/ml of Brefeldin A overnight disassembles the Golgi and causes the accumulation of procollagen (red) in the ER (green) and inhibits its transport to Golgi in AA-stimulated OBs. Scale bars, 10 µm. (D–E) Collagen immunoblots of control and AA-treated cell lysates and their conditioned media. The higher band on the blot represents the partially mature basal procollagen residing in the ER (as most of the procollagen modification occurs in ER) while the lower bands likely represent the cleaved procollagen peptides. The lower band appeared after AA addition presumably after processing of procollagen in the Golgi. Treatment of cells with BFA disrupted procollagen modifications in Golgi and therefore only the higher procollagen band was apparent in the immunoblot, presumably the procollagen in the ER. (E) BFA treatment also inhibited both basal and processed collagen secretion in the conditioned media collected from control and AA-stimulated cells as shown in the immunoblot.</p

Time-course of movement of procollagen in AA-differentiating MC3T3-E1 cells.

<p>Cells were fixed and immunostained for extracellular procollagen (in blue). The same cells were then permeabilized using Triton X-100 and were immunostained for internal procollagen as well as secreted collagen (both shown in red) since the antibody recognizes both procollagen and mature forms. The panels depict cells fixed at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours after AA addition. Intracellular procollagen was visible in the Golgi as early as 4 hours after AA stimulation and extracellular procollagen was visible at 6 hours. The 6 hour AA treatment was selected for further experiments as sufficient for procollagen translocation from ER to Golgi to the PM for secretion was observed at this timepoint. Scale bars, 10 µm.</p

Reduced extracellular collagen secretion levels in AA-stimulated MC3T3-E1 OBs expressing mutant Rab GTPases.

<p>Cells were plated on CYTOO coverslips for 1 hour, transfected with Rab1-DN-GFP (A), Rab3d-GFP (B) or Rab27b-GFP (C) for 9 hours during which they were differentiated with AA for 8 hours. Cells were then fixed and immunostained with extracellular collagen in cyan and total procollagen shown in red. Nuclei were stained with DAPI (blue). The dotted circles in black and white images point to extracellular collagen in transfected cells. Scale bars, 10 µm. (D–F) Graphs present the mean collagen intensity levels in cells expressing mutant Rabs for 9 hours compared to untransfected cells, which was quantified using ImageJ. Expression of all three mutant Rabs resulted in a significant reduction in extracellular collagen secretion. * p<0.05. All data are reported as mean ± SEM from 3 independent experiments (n = 50).</p