Leucine-rich Acidic Nuclear Protein Research Articles

Aging- and injury-related differential apoptotic response in the dentate gyrus of the hippocampus in rats following brain trauma

The elderly are among the most vulnerable to traumatic brain injury (TBI) with poor functional outcomes and impaired cognitive recovery. Of the pathological changes that occur following TBI, apoptosis is an important contributor to the secondary insults and subsequent morbidity associated with TBI. The current study investigated age-related differences in the apoptotic response to injury, which may represent a mechanistic underpinning of the heightened vulnerability of the aged brain to TBI. This study compared the degree of TBI-induced apoptotic response and changes of several apoptosis-related proteins in the hippocampal dentate gyrus (DG) of juvenile and aged animals following injury. Juvenile (p28) and aged rats (24 months) were subjected to a moderate fluid percussive injury or sham injury and sacrificed at 2 days post-injury. One group of rats in both ages was sacrificed and brain sections were processed for TUNEL and immunofluorescent labeling to assess the level of apoptosis and to identify cell types which undergo apoptosis. Another group of animals was subjected to proteomic analysis, whereby proteins from the ipsilateral DG were extracted and subjected to 2D-gel electrophoresis and mass spectrometry analysis. Histological studies revealed age- and injury-related differences in the number of TUNEL-labeled cells in the DG. In sham animals, juveniles displayed a higher number of TUNEL+ apoptotic cells located primarily in the subgranular zone of the DG as compared to the aged brain. These apoptotic cells expressed the early neuronal marker PSA-NCAM, suggestive of newly generated immature neurons. In contrast, aged rats had a significantly higher number of TUNEL+ cells following TBI than injured juveniles, which were NeuN-positive mature neurons located predominantly in the granule cell layer. Fluorescent triple labeling revealed that microglial cells were closely associated to the apoptotic cells. In concert with these cellular changes, proteomic studies revealed both age-associated and injury-induced changes in the expression levels of three apoptotic-related proteins: hippocalcin, leucine-rich acidic nuclear protein and heat shock protein 27. Taken together, this study revealed distinct apoptotic responses following TBI in the juvenile and aged brain which may contribute to the differential cognitive recovery observed.

LANP mediates neuritic pathology in Spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease that results from a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). Although the functions of ATXN1 are still largely unknown, there is evidence to suggest that ATXN1 plays a role in regulating gene expression, the earliest process known to go awry in SCA1 mouse models. In this study, we show that ATXN1 reduces histone acetylation, a post-translational modification of histones associated with enhanced transcription, and represses histone acetyl transferase-mediated transcription. In addition, we find that depleting the Leucine-rich Acidic Nuclear Protein (LANP)—an ATXN1 binding inhibitor of histone acetylation—reverses aspects of SCA1 neuritic pathology.

Production of Hepatopoietin Cn in High-Cell-Density Cultures of Recombinant Escherichia coli and Detection of its Antioxygen Activity

Hepatopoietin Cn (HPPCn), a member of the leucine-rich acidic nuclear protein (LANP) family, is a novel human hepatic growth factor that stimulates DNA synthesis and hepatocyte proliferation in vivo and in vitro by activating several signaling pathways. We performed pilot-scale production of recombinant human HPPCn (rhHPPCn) protein to elucidate its role in liver protection. An Escherichia coli BL21 (DE3) strain containing a pET-24a-HPPCn expression vector was cultivated in a 40-l bioreactor. High-cell-density fed-batch fermentation using this strain yielded a high concentration of rhHPPCn protein in the form of inclusion bodies. After washing and solubilization of the protein, rhHPPCn refolding was achieved by dilution of the denaturant. Then, the renatured protein was purified chromatographically. Assessment of the biological activity of the obtained protein showed that rhHPPCn could attenuate the oxidative stress in ethanol-induced hepatocyte injury. We successfully achieved large-scale production of biologically active rhHPPCn protein and obtained yields greater than 40mg rhHPPCn per gram dry cell weight by applying the above-mentioned renaturation and purification procedures. The obtained volumetric yield corresponds to 640mg of biologically active rhHPPCn per liter of culture broth. Additionally, the recombinant HPPCn protein was confirmed to protect hepatocytes against ethanol-induced oxidative stress.

CXCL12-Mediated Regulation of ANP32A/Lanp, A Component of the Inhibitor of Histone Acetyl Transferase (INHAT) Complex, in Cortical Neurons

The chemokine receptor CXCR4 and its endogenous ligand, CXCL12, are involved in development and homeostasis of the central nervous system and in the neuropathology of various neuroinflammatory/infectious disorders, including neuroAIDS. Our previous studies have shown that CXCR4 regulates cell cycle proteins that affect neuronal survival, such as the retinoblastoma protein, Rb. These studies also suggested that Rb-mediated gene repression might be involved in the neuronal protection against NMDA exitotoxicity conferred by stimulation of the CXCL12/CXCR4 axis. In order to further test this hypothesis, we focused on the potential interaction of Rb with another protein implicated in regulation of gene expression, leucine-rich acidic nuclear protein (Lanp), also known as ANP32A/pp32/PHAP1. Lanp is a critical member of the protein complex inhibitor of histone acetyl transferase (INHAT), which prevents histone tail's acetylation, thus leading to transcriptional repression. Our data show that, in primary rat cortical neurons cultured for up to 30 days, Lanp is predominantly localized into the nucleus throughout the culture period, in line with in vivo evidence. Moreover, co-immunoprecipitation experiments show that endogenous Lanp interacts with Rb in neurons. Stimulation of CXCR4 by its endogenous ligand, CXCL12, increased Lanp protein levels in these neurons. Importantly, the effect of CXCL12 was preserved after exposure of neurons to NMDA. Finally, overexpression of exogenous Lanp in the neurons protects them from excitotoxicity. Overall, these findings suggest that Lanp can interact with Rb in both young and mature neurons and is implicated in the regulation of neuronal survival by CXCL12/CXCR4.Electronic supplementary materialThe online version of this article (doi:10.1007/s11481-010-9228-5) contains supplementary material, which is available to authorized users.

Neuronal Differentiation Is Regulated by Leucine-rich Acidic Nuclear Protein (LANP), a Member of the Inhibitor of Histone Acetyltransferase Complex

Neuronal differentiation is a tightly regulated process characterized by temporal and spatial alterations in gene expression. A number of studies indicate a significant role for histone acetylation in the regulation of genes involved in development. Histone acetylation is regulated by histone deacetylases and histone acetyltransferases. Recent findings suggest that these catalytic activities, in turn, are modulated by yet another set of regulators. Of considerable interest in this context is the possible role of the INHAT (inhibitor of histone acetyltransferase) complex, comprised of a group of acidic proteins that suppress histone acetylation by a novel "histone-masking" mechanism. In this study, we specifically examined the role of the leucine-rich acidic nuclear protein (LANP), a defining member of the INHAT complex whose expression is tightly regulated in neuronal development. We report that depleting LANP in neuronal cell lines promotes neurite outgrowth by inducing changes in gene expression. In addition, we show that LANP directly regulates expression of the neurofilament light chain, an important neuron-specific cytoskeletal gene, by binding to the promoter of this gene and modulating histone acetylation levels. Finally, we corroborated our findings in vivo by demonstrating increased neurite outgrowth in primary neurons obtained from LANP null mice, which is also accompanied by increased histone acetylation at the NF-L promoter. Taken together, these results implicate INHATs as a distinct class of developmental regulators involved in the epigenetic modulation of neuronal differentiation.

The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats*

Hepatopoietin Cn (HPPCn) is a member of the leucine-rich acidic nuclear protein family (LANP), and studies of partially hepatectomized (PH) mice show that levels of HPPCn mRNA increase following liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. The aim of the study was to evaluate the protective effect of rhHPPCn on liver injury and fibrosis induced by carbon tetrachloride (CCl4) injection. Wistar rats weighing 200 g were given a single and repeated intraperitoneal injections of CCl4. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in rat serum were measured using biochemical assay. Hepatic hydroxyproline (Hyp) level was determined in the hydrolysates of liver samples. Immunostaining and Masson's trichrome staining were conducted to evaluate hepatocyte proliferation and fibrosis. The results showed that exogenous rhHPPCn could alleviate hepatocyte necrosis and protect the liver from the development of fibrotic lesions by proliferation stimulation. Additionally, HPPCn could reduce ALT/AST levels in rat serum following single and repeated CCl4 injection. It was suggested that HPPCn could protect hepatocytes from injury induced by CCl4 as a proliferation stimulator.

I PP2A 1 Affects Tau Phosphorylation via Association with the Catalytic Subunit of Protein Phosphatase 2A

In Alzheimer disease (AD) brain, the level of I (1)(PP2A), a 249-amino acid long endogenous inhibitor of protein phosphatase 2A (PP2A), is increased, the activity of the phosphatase is decreased, and the microtubule-associated protein Tau is abnormally hyperphosphorylated. However, little is known about the detailed regulatory mechanism by which PP2A activity is inhibited by I (1)(PP2A) and the consequent events in mammalian cells. In this study, we found that both I (1)(PP2A) and its N-terminal half I (1)(PP2A(1-120)), but neither I (1)(PP2A(1-163)) nor I (1)(PP2A(164-249)), inhibited PP2A activity in vitro, suggesting an autoinhibition by amino acid residues 121-163 and its neutralization by the C-terminal region. Furthermore, transfection of NIH3T3 cells produced a dose-dependent inhibition of PP2A activity by I (1)(PP2A)(1). I (PP2A) and PP2A were found to colocalize in PC12 cells. I (1)(PP2A) could only interact with the catalytic subunit of PP2A (PP2Ac) and had no interaction with the regulatory subunits of PP2A (PP2A-A or PP2A-B) using a glutathione S-transferase-pulldown assay. The interaction was further confirmed by coimmunoprecipitation of I (1)(PP2A) and PP2Ac from lysates of transiently transfected NIH3T3 cells. The N-terminal isotype specific region of I (1)(PP2A) was required for its association with PP2Ac as well as PP2A inhibition. In addition, the phosphorylation of Tau was significantly increased in PC12/Tau441 cells transiently transfected with full-length I (1)(PP2A) and with PP2Ac-interacting I (1)(PP2A) deletion mutant 1-120 (I (1)(PP2A)DeltaC2). Double immunofluorescence staining showed that I (1)(PP2A) and I (1)(PP2A)DeltaC2 increased Tau phosphorylation and impaired the microtubule network and neurite outgrowth in PC12 cells treated with nerve growth factor.

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn

Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.

The role of LANP and ataxin 1 in E4F‐mediated transcriptional repression

The leucine-rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1--a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)--we tested whether ataxin 1 can alter the E4F-LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP-E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1.

Generation and Characterization of LANP/pp32 Null Mice

The leucine-rich acidic nuclear protein (LANP) belongs to a family of evolutionarily conserved proteins that are characterized by an amino-terminal domain rich in leucine residues followed by a carboxy-terminal acidic tail. LANP has been implicated in the regulation of a variety of cellular processes including RNA transport, transcription, apoptosis, vesicular trafficking, and intracellular signaling. Abundantly expressed in the developing cerebellum, this protein has also been hypothesized to play a role in cerebellar morphogenesis. LANP has been implicated in disease biology as well, both as a mediator of toxicity in spinocerebellar ataxia type 1 and as a tumor suppressor in cancers of the breast and prostate. To better understand the function of this multifaceted protein, we have generated mice lacking LANP. Surprisingly, these mice are viable and fertile. In addition we could not discern any derangements in any of the major organ systems, including the nervous system, which we have studied in detail. Overall our results point to a functional redundancy of LANP's function, most likely provided by its closely related family members.

Mapmodulin/Leucine-rich Acidic Nuclear Protein Binds the Light Chain of Microtubule-associated Protein 1B and Modulates Neuritogenesis

We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins.

Crystal structure of the apoptosis-inducing human granzyme A dimer.

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 A, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor-plasmin and cofactor-plasminogen complexes.

Molecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain

Leucine-rich acidic nuclear protein (LANP) is a member of the leucine-rich repeats (LRRs) superfamily. Here we report on a human homologue of LANP, encoded by the gene ANP32E (alias LANPL). The gene was cloned and identified during large-scale sequencing analysis of a human fetal brain cDNA library. The human protein shared 70% amino acid identity with rat LANP. According to bioinformatics analysis, ANP32E is located on chromosome 1q22. RT-PCR analysis indicates that ANP32E was expressed in human peripheral blood leukocytes, colon, small intestine, prostate, thymus, spleen, skeletal muscle, liver and kidney.

Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein

We previously reported that the nuclear localization signal (NLS) peptides stimulate the in vitro phosphorylation of several proteins, including a 34 kDa protein. In this study, we show that this specific 34 kDa protein is a novel murine leucine-rich acidic nuclear protein (LANP)-like large protein (mLANP-L). mLANP-L was found to have a basic type NLS. The co-injection of Q69LRan-GTP or SV40 T-antigen NLS peptides prevented the nuclear import of mLANP-L. mLANP-L NLS bound preferentially to Rch1 and NPI-1, but not to the Qip1 subfamily of importin α. These findings suggest that mLANP-L is transported into the nucleus by Rch1 and/or NPI-1.

Progress in pathogenesis studies of spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited disorder characterized by progressive loss of coordination, motor impairment and the degeneration of cerebellar Purkinje cells, spinocerebellar tracts and brainstem nuclei. Many dominantly inherited neurodegenerative diseases share the mutational basis of SCA1: the expansion of a translated CAG repeat coding for glutamine. Mice lacking ataxin-1 display learning deficits and altered hippocampal synaptic plasticity but none of the abnormalities seen in human SCA1; mice expressing ataxin-1 with an expanded CAG tract (82 glutamine residues), however, develop Purkinje cell pathology and ataxia. These results suggest that mutant ataxin-1 gains a novel function that leads to neuronal degeneration. This novel function might involve aberrant interaction(s) with cell-specific protein(s), which in turn might explain the selective neuronal pathology. Mutant ataxin-1 interacts preferentially with a leucine-rich acidic nuclear protein that is abundantly expressed in cerebellar Purkinje cells and other brain regions affected in SCA1. Immunolocalization studies in affected neurons of patients and SCA1 transgenic mice showed that mutant ataxin-1 localizes to a single, ubiquitin-positive nuclear inclusion (NI) that alters the distribution of the proteasome and certain chaperones. Further analysis of NIs in transfected HeLa cells established that the proteasome and chaperone proteins co-localize with ataxin-1 aggregates. Moreover, overexpression of the chaperone HDJ-2/HSDJ in HeLa cells decreased ataxin-1 aggregation, suggesting that protein misfolding might underlie NI formation. To assess the importance of the nuclear localization of ataxin-1 and its role in SCA1 pathogenesis, two lines of transgenic mice were generated. In the first line, the nuclear localization signal was mutated so that full-length mutant ataxin-1 would remain in the cytoplasm; mice from this line did not develop any ataxia or pathology. This suggests that mutant ataxin-1 is pathogenic only in the nucleus. To assess the role of the aggregates, transgenic mice were generated with mutant ataxin-1 without the self-association domain (SAD) essential for aggregate formation. These mice developed ataxia and Purkinje cell abnormalities similar to those seen in SCA1 transgenic mice carrying full-length mutant ataxin-1, but lacked NIs. The nuclear milieu is thus a critical factor in SCA1 pathogenesis, but large NIs are not needed to initiate pathogenesis. They might instead be downstream of the primary pathogenic steps. Given the accumulated evidence, we propose the following model for SCA1 pathogenesis: expansion of the polyglutamine tract alters the conformation of ataxin-1, causing it to misfold. This in turn leads to aberrant protein interactions. Cell specificity is determined by the cell-specific proteins interacting with ataxin-1. Submicroscopic protein aggregation might occur because of protein misfolding, and those aggregates become detectable as NIs as the disease advances. Proteasome redistribution to the NI might contribute to disease progression by disturbing proteolysis and subsequent vital cellular functions.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

Protein misfolding is now recognized to be a central feature of neurodegenerative diseases. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, and the polyglutamine (polygln) diseases all represent proteinopathies—diseases in which a particular protein or set of proteins misfolds and aggregates. In the past decade, mutations have been identified in an increasing number of genes underlying specific forms of neurodegeneration—for example, genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 in AD, parkin and alpha-synuclein in PD, Cu/Zn superoxide dismutase in ALS, tau in frontotemporal dementia, prion protein in Creutzfeldt-Jacob and Gerstmann-Straussler diseases, and at least eight different proteins in the polygln diseases (Hardy and GwinnHardy 1998; Price et al. 1998). Identification of these mutations was a first step in understanding the disease process; next, it will be necessary to explain how specific changes in disease proteins may alter the neuron’s physiology and to further question how neuronal responses to the mutant protein influence the disease process. This review examines the fate of mutant protein in neurons and the potential role of posttranslational events in pathogenesis, with particular focus on the polygln diseases. Posttranslational events have already been implicated

The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. SCA1 belongs to a growing group of neurodegenerative disorders caused by expansion of CAG repeats, which encode glutamine. Although the proteins containing these repeats are widely expressed, the neurodegeneration in SCA1 and other polyglutamine diseases selectively involves a few neuronal subtypes. The mechanism(s) underlying this neuronal specificity is unknown. Here we show that the cerebellar leucine-rich acidic nuclear protein (LANP) interacts with ataxin-1, the SCA1 gene product. LANP is expressed predominantly in Purkinje cells, the primary site of pathology in SCA1. The interaction between LANP and ataxin-1 is significantly stronger when the number of glutamines is increased. Immunofluorescence studies demonstrate that both LANP and ataxin-1 colocalize in nuclear matrix-associated subnuclear structures. The features of the interaction between ataxin-1 and LANP, their spatial and temporal patterns of expression, and the colocalization studies indicate that cerebellar LANP is involved in the pathogenesis of SCA1.

A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.

A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons.