Rationale: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. Objective: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. Methods and Results: Ex vivo perfused HKII؉/؊ hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemiareperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 mol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. Conclusions:Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential. (Circ Res. 2011;108:1165-1169.)

This study identifies a novel therapeutic strategy against cisplatin-resistant lung squamous cell carcinoma (LSCC) using mouse models and patient samples. LSCC chemoresistance depends on LUBAC and high NF-κB activity, mechanisms that can be targeted to increase therapy response.

Summary N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser‐dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide‐laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring.

The endocytosis of LDL via the LDL receptor (LDLR) has served as a paradigm for receptor-mediated endocytosis since its initial description by the Brown and Goldstein laboratory ( 1 ). At the plasma membrane, LDLR binds extracellular LDL and is endocytosed by clathrindependent mechanisms in clathrin-coated pits. Acidification of early endocytic vesicles liberates LDL from the receptor and allows the cargo to be delivered to lysosomes where the lipoprotein particle is degraded and cholesterol is salvaged for cellular use. In contrast, the LDLR recycles back to the plasma membrane and can be reused in a new round of endocytosis ( 2 ). Clathrin is not suffi cient to support LDLR internalization. Effi cient internalization of the LDLR requires adaptor proteins that tether the receptor to the endocytic machinery. The best-described adaptor proteins are the autosomal recessive hypercholesterolemia (ARH) and disabled 2 (DAB2) ( 3, 4 ). These adaptors interact with AP2 and clathrin and in parallel via their phospho-tyrosine binding domain with the NPxY endocytosis motif present in the intracellular tail of the LDLR ( 5-7 ). Michaely et al. ( 8 ) recently reported that a HIC sequence-motif present downstream of the canonical NPxY motif can also be used for clathrin-dependent uptake of ␤ -VLDL, thus representing a second clathrin-dependent route for LDLR internalization. The clathrin-mediated endocytic strategy is evolutionarily conserved , and is used by different Abstract Low density lipoprotein (LDL) cholesterol is taken up into cells via clathrin-mediated endocytosis of the LDL receptor (LDLR). Following dissociation of the LDLR-LDL complex, LDL is directed to lysosomes whereas the LDLR recycles to the plasma membrane. Activation of the sterol-sensing nuclear receptors liver X receptors (LXRs) enhances degradation of the LDLR . This depends on the LXR target gene inducible degrader of the LDLR (IDOL), an E3-ubiquitin ligase that promotes ubiquitylation and lysosomal degradation of the LDLR. How ubiquitylation of the LDLR by IDOL controls its endocytic traffi cking is currentlyunknown. Using genetic-and pharmacological-based approaches coupled to functional assessment of LDL uptake, we show that the LXR-IDOL axis targets a LDLR pool present in lipid rafts. IDOL-dependent internalization of the LDLR is independent of clathrin, caveolin, macroautophagy, and dynamin. Rather, it depends on the endocytic protein epsin. Consistent with LDLR ubiquitylation acting as a sorting signal, degradation of the receptor can be blocked by perturbing the endosomal sorting complex required for transport (ESCRT) or by USP8, a deubiquitylase implicated in sorting ubiquitylated cargo to multivesicular bodies. In summary, we provide evidence for the existence of an LXR-IDOL-mediated internalization pathway for the LDLR that is distinct from that used for lipoprotein uptake. -Sorrentino, V., J. K. Nelson, E. Maspero, A. R. A. Marques, L. Scheer, S. Polo, and N. Zelcer. The LXR-IDOL axis defi nes a clathrin-, caveolae-, and dynamin-independe...

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T he (pro)renin receptor [(P)RR] has been implicated as a receptor for renin/prorenin (denoted as [pro]renin)-stimulated signaling, and plays a role in local renin-angiotensin system activation by nonproteolytically activating bound prorenin. 1 In experimental assays (pro)renin-(P)RR signaling results in extracellular signal-regulated kinase 1/2 (Erk1/2) activation, and as a consequence upregulation of profibrotic factors, such as transforming growth factor β, collagen, and fibronectin. [2][3][4][5][6] However, the physiological relevance of the (pro)renin-(P)RR interaction is questionable because the (pro)renin concentrations required are >1000× higher than observed under (patho) physiological conditions. 7,8 Recently, (pro)renin-independent functions for (P)RR have been reported, including a function as an accessory protein of the vacuolar H + -ATPase (V-ATPase). 9 V-ATPases are multisubunit complexes, and they are expressed virtually in all cells types. They play an important role in protein trafficking, receptor recycling, and lysosomal degradation by acidifying intracellular compartments.10,11 Depletion of the (P)RR results in decreased protein levels of V-ATPase subunits, impaired acidification of intracellular compartments, and defects in autophagy.12-14 V-ATPases are also found at the plasma membrane in certain cell types, such as intercalated cells of the collecting duct. Accordingly, we previously reported that the (P)RR is required for both prorenin-dependent and proreninindependent regulation of V-ATPase activity in collecting duct cells.15 The (P)RR has been also recently implicated in canonical Wnt and PCP signaling, [16][17][18] emphasizing the notion that our understanding of (P)RR function remains incomplete. In This Issue, see p 183Editorial, see p 187To address this, we used an unbiased proteomics approach to discover potential novel functions of the (P)RR. We mapped the (P)RR-interactome and identified sortilin 1 (SORT1) as Objective: To uncover renin-angiotensin system-independent functions of the (P)RR. Methods and Results: We used a proteomics-based approach to purify and identify (P)RR-interacting proteins.This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by coimmunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (

Our results support the notion that IDOL contributes to variation in circulating levels of LDL-C. Strategies to inhibit IDOL activity may therefore provide a novel therapeutic venue to treating dyslipidaemia.