Interaction of DISC1 with the PTB domain of Tensin2

The gene for Disrupted-in-Schizophrenia 1 (DISC1) is amongst the most significant risk genes for schizophrenia. The DISC1 protein is an intracellular scaffolding molecule thought to act an important hub for protein interactions involved in signalling for neural cell differentiation and function. Tensin2 is an intracellular actin-binding protein that bridges the intracellular portion of transmembrane receptors to the cytoskeleton, thereby regulating signalling for cell shape and motility. In this study, we probed in molecular detail a novel interaction between DISC1 and Tensin2. Western blot and confocal microscopic analyses revealed widespread expression of both DISC1 and Tensin2 proteins throughout the mouse brain. Furthermore, we have developed novel anti-DISC1 antibodies that verified the predominant expression of a 105-kDa isoform of DISC1 in the rodent brain as well as in human cells. In the mouse brain, both proteins showed region-specific expression patterns, including strong expression in the pyramidal cell layer of the hippocampus and dentate gyrus. DISC1–Tensin2 colocalisation was most clearly observed in the Purkinje cells of the mouse cerebellum. Biochemical coimmunoprecipitation experiments revealed an interaction between endogenous DISC1 and Tensin2 proteins in the mouse brain. Further pulldown studies in human cells using Myc-tagged Tensin2 constructs revealed that DISC1 specifically interacts with the C-terminal PTB domain of Tensin2 in a phosphorylation-independent manner. This new knowledge on the DISC1–Tensin2 interaction, as part of the wider DISC1 interactome, should further elucidate the signalling pathways that are perturbed in schizophrenia and other mental disorders.     

Chromosome boundary elements and regulation of heterochromatin spreading

Chromatin is generally classified as euchromatin or heterochromatin, each with distinct histone modifications, compaction levels, and gene expression patterns. Although the proper formation of heterochromatin is essential for maintaining genome integrity and regulating gene expression, heterochromatin can also spread into neighboring regions in a sequence-independent manner, leading to the inactivation of genes. Because the distance of heterochromatin spreading is stochastic, the formation of boundaries, which block the spreading of heterochromatin, is critical for maintaining stable gene expression patterns. Here we review the current understanding of the mechanisms underlying heterochromatin spreading and boundary formation.     

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2^+/+ and CerS2^?/? mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2^?/? mice developed more severe disease than CerS2^+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2^?/? mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2^?/? mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2^?/? mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4⁺ and Th17⁺ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.     

Silencing of RB1 but not of RB2/P130 induces cellular senescence and impairs the differentiation potential of human mesenchymal stem cells

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21^cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.     

Nicotinamide is an inhibitor of SIRT1 in vitro,but can be a stimulator in cells

Nicotinamide (NAM), a form of vitamin B₃, plays essential roles in cell physiology through facilitating NAD⁺ redox homeostasis and providing NAD⁺ as a substrate to a class of enzymes that catalyze non-redox reactions. These non-redox enzymes include the sirtuin family proteins which deacetylate target proteins while cleaving NAD⁺ to yield NAM. Since the finding that NAM exerts feedback inhibition to the sirtuin reactions, NAM has been widely used as an inhibitor in the studies where SIRT1, a key member of sirtuins, may have a role in certain cell physiology. However, once administered to cells, NAM is rapidly converted to NAD⁺ and, therefore, the cellular concentration of NAM decreases rapidly while that of NAD⁺ increases. The result would be an inhibition of SIRT1 for a limited duration, followed by an increase in the activity. This possibility raises a concern on the validity of the interpretation of the results in the studies that use NAM as a SIRT1 inhibitor. To understand better the effects of cellular administration of NAM, we reviewed published literature in which treatment with NAM was used to inhibit SIRT1 and found that the expected inhibitory effect of NAM was either unreliable or muted in many cases. In addition, studies demonstrated NAM administration stimulates SIRT1 activity and improves the functions of cells and organs. To determine if NAM administration can generate conditions in cells and tissues that are stimulatory to SIRT1, the changes in the cellular levels of NAM and NAD⁺ reported in the literature were examined and the factors that are involved in the availability of NAD⁺ to SIRT1 were evaluated. We conclude that NAM treatment can hypothetically be stimulatory to SIRT1.     

Inhibition of proliferation by 1-8U in interferon-α-responsive and non-responsive cell lines

Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of 1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction. Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation. Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development. Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003 RID="*" ID="*"Corresponding author.     

A central role for calcineurin in protein misfolding neurodegenerative diseases

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca²⁺) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca²⁺ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca²⁺ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca²⁺/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.     

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function

ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560–563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560–563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE–ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560–563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [³H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.     

Heterochromatin associated with active versus inactive centromeres of mouse replicates at different times

Summary A subline of mouse L-cells carries a dicentric chromosome in which one centromere always separates prematurely. This centromere is not involved in the dynamics of chromosome migration and is considered inactive. By use of anti-BRdU antibody binding to BRdU-treated chromosomes it is shown that the pericentric constitutive heterochromatin associated with the prematurely separating centromere replicates earlier than its counterpart associated with the active centromere and even before several euchromatic regions in the genome. These results point to a possible mechanism by which dicentric chromosomes segregate equationally.     

Heterochromatin associated with active versus inactive centromeres of mouse replicates at different times

A subline of mouse L-cells carries a dicentric chromosome in which one centromere always separates prematurely. This centromere is not involved in the dynamics of chromosome migration and is considered inactive. By use of anti-BRdU antibody binding to BRdU-treated chromosomes it is shown that the pericentric constitutive heterochromatin associated with the prematurely separating centromere replicates earlier than its counterpart associated with the active centromere and even before several euchromatic regions in the genome. These results point to a possible mechanism by which dicentric chromosomes segregate equationally.     

The regulation of embryonic patterning and DNA replication by geminin

Geminin is a multifunctional protein. After DNA replication is initiated during a cell cycle, geminin binds to Cdt1, one of the key DNA replication licensing factors. This highly regulated interaction sequestrates Cdt1, thus preventing DNA rereplication in the same cell cycle. In addition, geminin directly interacts with Six3 and Hox homeodomain proteins during embryogenesis and inhibits their functions. The regulation of Hox function by geminin also involves a transient association with the Hox repressive Polycomb complex. The functions of geminin to obstruct key molecules of both cell proliferation and embryonic development suggest a competitive coordination of these two processes.Received 10 December 2004; received after revision 27 January 2005; accepted March 2005     

Sumoylation regulates diverse biological processes

Ten years after its discovery, the small ubiquitin-like protein modifier (SUMO) has emerged as a key regulator of proteins. While early studies indicated that sumoylation takes place mainly in the nucleus, an increasing number of non-nuclear substrates have recently been identified, suggesting a wider stage for sumoylation in the cell. Unlike ubiquitylation, which primarily targets a substrate for degradation, sumoylation regulates a substrate’s functions mainly by altering the intracellular localization, protein-protein interactions or other types of post-translational modifications. These changes in turn affect gene expression, genomic and chromosomal stability and integrity, and signal transduction. Sumoylation is counter-balanced by desumoylation, and well-balanced sumoylation is essential for normal cellular behaviors. Loss of the balance has been associated with a number of diseases. This paper reviews recent progress in the study of SUMO pathways, substrates, and cellular functions and highlights important findings that have accelerated advances in this study field and link sumoylation to human diseases. Received 19 March 2007; received after version 16 July 2007; accepted 1 August 2007     

Transgenic animal models of sickle cell disease

An animal model which allows study of chronic processes occurring in sickle cell disease has finally been realized with the development of several lines of transgenic mice which express high levels of ^s or ^s-variants in their red cells. The red cells of all mouse lines exhibit characteristic sickle shapes on deoxygenation and most lines have enlarged spleens and mildly elevated reticulocytes in adult mice; both of these properties are suggestive of enhanced red cell destruction and erythropoiesis. However, all lines examined to date have normal hemoglobin levels in adult mice. In one mouse line under ambient conditions, retinopathy and abnormal renal function have been observed and in the same line under hypoxic conditions, anemia, irreversibly sickled cell formation, and urine concentrating defect have been observed. The current mouse lines will allow some aspects of sickle cell disease to be studied, but sigificant improvements can still be made in the transgenic mouse model.     

Primary cilia proteins: ciliary and extraciliary sites and functions

Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these “ciliary” proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes’ influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of “cilia” proteins as a means to demonstrate the potential non-ciliary roles for these proteins.     

DNG1, a Dictyostelium homologue of tumor suppressor ING1 regulates differentiation of Dictyostelium cells

dng1 is a Dictyostelium homologue of the mammalian tumor suppressor ING gene. DNG1 protein localizes in the nucleus, and has a highly conserved PHD finger domain found in chromatin-remodeling proteins. Both dng1 disruption and overexpression impaired cell proliferation. In dng1-null cells, the progression of differentiation was delayed in a cell-density-dependent manner, and many tiny aggregates were formed. Exogenously applied cAMP pulses reversed the inhibitory effect caused by dng1 disruption on the aggregation during early development, but formation of tiny aggregates was not restored. dng1-overexpressing cells acquired the ability to undergo chemotaxis to cAMP earlier and exhibited enhanced differentiation. These phenotypes were found to be coupled with altered expressions of early genes such as cAMP receptor 1 (car1) and contact site A (csA). Furthermore, disordered histone modifications were demonstrated in dng1-null cells. These results suggest a regulatory role of dng1 in the transition of cells from growth to differentiation.Received 29 December 2004; received after revision 24 May 2005; accepted 26 May 2005     

Giant axonal neuropathy

Giant axonal neuropathy (GAN) is a rare autosomal recessive disorder affecting both the central and peripheral nervous systems. Cytopathologically, the disorder is characterized by giant axons with derangements of cytoskeletal components. Geneticists refined the chromosomal interval containing the locus, culminating in the cloning of the defective gene, GAN. To date, many distinct mutations scattered throughout the coding region of the locus have been reported by researchers from different groups around the world. GAN encodes the protein, gigaxonin. Recently, a genetic mouse model of the disease was generated by targeted disruption of the locus. Over the years, the molecular mechanisms underlying GAN have attracted much interest. Studies have revealed that gigaxonin appears to play an important role in cytoskeletal functions and dynamics by directing ubiquitin-mediated degradations of cytoskeletal proteins. Aberrant accumulations of cytoskeletal-associated proteins caused by a defect in the ubiquitinproteasome system (UPS) have been shown to be responsible for neurodegeneration occurring in GAN-null neurons, providing strong support for the notion that UPS plays crucial roles in cytoskeletal functions and dynamics. However, many key questions about the disease remain unanswered. Received 6 September 2006; received after revision 11 October 2006; accepted 5 December 2006 Y. Yang, E. Allen The authors contributed equally to this work.