OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)—lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3β(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER–LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER–LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281–1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007     

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds α-tubulin and promotes α/β dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds α-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Δ mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Δ mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.     

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K_ATP) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K_ATP channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K_ATP-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.     

Synphilin-1 inhibits alpha-synuclein degradation by the proteasome

Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson’s disease. Protein–protein interactions are critical in the regulation of cell proteostasis. Synphilin-1 interacts both in vitro and in vivo with alpha-synuclein promoting its aggregation. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense mutants by the 20S proteasome due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (amino acids 331–555) with the N-terminal region (amino acids 1–60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. Synphilin-1 inhibition can be relieved by co-expression of Siah-1 that targets synphilin-1 to degradation. Synphilin-1 inhibition of the proteasomal pathway of degradation of alpha-synuclein may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.     

The actin-binding domain of actin filament-associated protein (AFAP) is involved in the regulation of cytoskeletal structure

Actin filament-associated protein (AFAP) plays a critical role in the regulation of actin filament integrity, formation and maintenance of the actin network, function of focal contacts, and cell migration. Here, we show that endogenous AFAP was present not only in the cytoskeletal but also in the cytosolic fraction. Depolymerization of actin filaments with cytochalasin D or latrunculin A increased AFAP in the cytosolic fraction. AFAP harbors an actin-binding domain (ABD) in its C-terminus. AFAPΔABD, an AFAP mutant with selective ABD deletion, was mainly in the cytosolic fraction when overexpressed in the cells, which was associated with a disorganized cytoskeleton with reduced stress fibers, accumulation of F-actin on cellular membrane, and formation of actin-rich small dots. Cortactin, a well-known podosome marker, was colocalized with AFAPΔABD in these small dots at the ventral surface of the cell, indicating that these small dots fulfill certain criteria of podosomes. However, these podosome-like small dots did not digest gelatin matrix. This may be due to the reduced interaction between AFAPΔABD and c-Src. When AFAPΔABD-transfected cells were stimulated with phorbol ester, they formed podosome-like structures with larger sizes, less numerous and longer life span, in comparison with wild-type AFAP-transfected cells. These results indicate that the association of AFAP with F-actin through ABD is crucial for AFAP to regulate cytoskeletal structures. The AFAPΔABD, as cytosolic proteins, may be more accessible to the cellular membrane, podosome-like structures, and thus be more interactive for the regulation of cellular functions.     

Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP–VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP–VAP association, alters the subcellular targeting of ORP–VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP–VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER–lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP–VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.     

Liver X receptors in cardiovascular and metabolic disease

Liver X receptors (LXRs) α and β are nuclear oxysterol receptors and metabolic sensors initially found to regulate cholesterol metabolism and lipid biosynthesis. Recent studies have elucidated the importance of LXR in the development of cardiovascular diseases and metabolic disorders. LXR agonists prevent development of atherosclerosis by modulation of metabolic as well as inflammatory gene expression in rodent models. Moreover, LXR activation inhibits hepatic gluconeogenesis and lowers serum glucose levels, indicating possible application of LXR activation in the treatment of diabetes mellitus. However, first-generation LXR agonists elevate hepatic and serum trigylceride levels, making subtype-specific agonists and selective LXR modulators rather than unselective LXR agonists a potential pharmacological strategy. This review summarizes the multiple physiological and pathophysiological implications of LXRs and observations that identify LXRs as potential targets for therapeutic interventions in human cardiovascular and metabolic disease. Received 30 August 2005; received after revision 10 October 2005; accepted 4 November 2005     

Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less ρ⁰ cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2α constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells. Independent of AMPK and mTOR, eIF-2α is constitutively phosphorylated in mutant cells, likely a signature of endoplasmic reticulum (ER)-stress response induced by the loss of oxidative phosphorylation. While the AMPK/eEF-2K/eEF-2 pathway appears to function in adaptation to physiological fluctuations in ATP levels in the mutant cells, the ER stress signified by constitutive protein synthesis inhibition through eIF-2α-mediated repression of translation initiation may have pathobiochemical consequences. Received 29 October 2008; received after revision 11 December 2008; accepted 16 December 2008     

Endoplasmic reticulum stress responses

In homeostasis, cellular processes are in a dynamic equilibrium. Perturbation of homeostasis causes stress. In this review I summarize how perturbation of three major functions of the endoplasmic reticulum (ER) in eukaryotic cells–protein folding, lipid and sterol biosynthesis, and storing intracellular Ca²⁺ – causes ER stress and activates signaling pathways collectively termed the unfolded protein response (UPR). I discuss how the UPR reestablishes homeostasis, and summarize our current understanding of how the transition from protective to apoptotic UPR signaling is controlled, and how the UPR induces inflammatory signaling. Received 21 August 2007; received after revision 26 October 2007; accepted 29 October 2007     

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1–212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1–212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1–3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region. Received 24 April 2007; received after revision 11 June 2007; accepted 13 June 2007     

Early Influences on Probability and Statistics in the Russian Empire

Historiography of the development of probability and statistics in the Russian Empire focusses on the contributions of the central figure Pafnutiy Lvovich Chebyshev (1821–1894) and his successors. The purpose of this article is to concentrate on an earlier period which culminates with Chebyshev, and specifically on two less-than-well-explored aspects: (1) The background and motivation for his activity in probability and statistics; (2) The French connections and influences on his work. The key figures in this account are A.F. Pavlovsky (1789–1875); M.V. Ostrogradsky (1801–1862); V.Y. Buniakovsky (1804–1889); N.D. Brashman (1796–1866); N.E. Zernov (1804–1862), S.G. Stroganov (1794–1882); P.S. de Laplace (1749–1827); A.L. Cauchy (1789–1857); I.J. Bienaymé (1796–1878); N.V. Khanykov (1819–1878); and, in Chebyshevs childhood, a governess and relation, Avdotiia Kvintillianovna Sukhareva. Chief among these, from the years of Chebyshevs maturity, are Buniakovsky and Bienaymé. The cultural contacts between France and the Russian Empire in the 19th century were strong, and these connections are particularly well-illustrated in this setting. (Received January 10, 1998)     

L1CAM malfunction in the nervous system and human carcinomas

Research over the last 25 years on the cell adhesion molecule L1 has revealed its pivotal role in nervous system function. Mutations of the human L1CAM gene have been shown to cause neurodevelopmental disorders such as X-linked hydrocephalus, spastic paraplegia and mental retardation. Impaired L1 function has been also implicated in the aetiology of fetal alcohol spectrum disorders, defective enteric nervous system development and malformations of the renal system. Importantly, aberrant expression of L1 has emerged as a critical factor in the development of human carcinomas, where it enhances cell proliferation, motility and chemoresistance. This discovery promoted collaborative work between tumour biologists and neurobiologists, which has led to a substantial expansion of the basic knowledge about L1 function and regulation. Here we provide an overview of the pathological conditions caused by L1 malfunction. We further discuss how the available data on gene regulation, molecular interactions and posttranslational processing of L1 may contribute to a better understanding of associated neurological and cancerous diseases.     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Homing endonucleases: from basics to therapeutic applications

Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12–40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.     

Heat shock protein 60: regulatory role on innate immune cells

Human heat shock protein 60 (Hsp60) exhibits immunoregulatory properties, primarily by inducing pro-inflammatory responses in innate immune cells. Extensive analyses identified specific receptor structures for the interaction of Hsp60 with these cells. The existence of distinct receptor structures responsible for Hsp60 binding and for Hsp60-induced release of pro-inflammatory mediators has been demonstrated, implying that the interaction of Hsp60 with innate immune cells is a multifaceted process. Distinct Hsp60 epitopes responsible for binding to innate immune cells and for the activation of these cells have been identified. Depending on the cell-type, the amino acid (aa) region 481–500 or the regions aa241–260, aa391–410 and aa461–480 are involved in Hsp60-binding to innate immune cells. An entirely different Hsp60-region, aa354–365 was found to bind lipopolysaccharide, thereby mediating the pro-inflammatory effects of Hsp60. Because of its immunoregulatory properties, Hsp60 has been proposed to act as intercellular danger signal, controlling innate and adaptive immune reactions. Received 19 September 2006; received after revision 13 October 2006; accepted 13 December 2006