Modulation of protein kinases and phosphoprotein phosphatases by a small acidic protein from bovine brains

Summary A small, acidic and heat-stable protein was purified from bovine brains by column chromatography on DEAE-cellulose, Bio-Gel HTP, Affi-Gel phenothiazine and Sephadex G-75. This protein stimulates megamodulin-dependent protein kinase I from brains and phosphoprotein phosphatases from either brain or yeast. However, it inhibits cyclic AMP-dependent protein kinases from skeletal muscle.Acknowledgments. This work was supported by a grant (RR-08229) from the National Institutes of Health, USA. W.N. Kuo is a recipient of a Distinguished Faculty Scholar Award from United Negro College Fund, Inc., USA.     

Pyridine as an unmasking reagent for lipoprotein complexes in the nervous system of protein deficient squirrel monkeys

Summary Acid hematin test with pyridine and Sudan black B controls was employed on selected areas of the brains of 115, 140 days fetuses, neonates and adult squirrel monkeys maintained on low and high protein diet. Our histochemical findings indicate that the reduction of phospholipids in the low protein fetuses and neonates is related to myelination, whereas in the adults, most of the lipids are bound to proteins and/or cerebrosides to form complexes, as revealed by the unmasking action of pyridine.Acknowledgements. This work was supported by U.S. PHS grants RR-00165 HD-06087 from National Institute of Health.     

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     

Neurodegeneration changes in primary central nervous system neurons transfected with the Alzheimer-associated neuronal thread protein gene

The AD7c-NTP gene is over-expressed in brains with Alzheimer's disease (AD), and increased levels of the corresponding protein are detectable in cortical neurons, brain tissue extracts, cerebrospinal fluid, and urine beginning early in the course of AD neurodegeneration. In the present study, we utilized a novel method to transfect post-mitotic primary neuronal cell cultures, and demonstrated that over-expression of the AD7c-NTP gene causes cell death and neuritic sprouting, two prominent abnormalities associated with AD. These results provide further evidence that aberrantly increas-ed AD7c-NTP expression may have a role in AD-type neurodegeneration. In addition, we demonstrate that primary post-mitotic neurons can be efficiently transfected with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in relevant in vitro models. Received 31 January 2001; received after revision 31 March 2001; accepted 4 April 2001     

Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons

Amyloid beta peptide (Aβ), the main component of senile plaques of Alzheimer’s disease brains, is produced by sequential cleavage of amyloid precursor protein (APP) and of its C-terminal fragments (CTFs). An unanswered question is how amyloidogenic peptides spread throughout the brain during the course of the disease. Here, we show that small lipid vesicles called exosomes, secreted in the extracellular milieu by cortical neurons, carry endogenous APP and are strikingly enriched in CTF-α and the newly characterized CTF-η. Exosomes from N2a cells expressing human APP with the autosomal dominant Swedish mutation contain Aβ peptides as well as CTF-α and CTF-η, while those from cells expressing the non-mutated form of APP only contain CTF-α and CTF-η. APP and CTFs are sorted into a subset of exosomes which lack the tetraspanin CD63 and specifically bind to dendrites of neurons, unlike exosomes carrying CD63 which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells, allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.     

Mapping the antigenicity of copper-treated cellular prion protein with the scrapie isoform

When recombinant and cellular prion protein (PrP(C)) binds copper, it acquires properties resembling the scrapie isoform (PrP(Sc)), namely protease resistance, detergent insolubility and increased beta sheet content. However, whether the conformations of PrP(C) induced by copper and PrP(Sc) are similar has not been studied in great detail. Here, we use a panel of seven monoclonal antibodies to decipher the epitopes on full-length mouse PrP(C) that are affected by exogenous copper, and to compare the antigenicity of the copper-treated full-length PrP(C) with the full-length PrP(Sc) present in scrapie-infected mouse brains. In the presence of copper, we found that epitopes along residues 115-130 and 153-165 become more accessible on PrP(C). These regions correspond to the two beta sheet strands in recombinant PrP and they were proposed to be important for prion conversion. However, when we compared the antibody-binding patterns between full-length PrP(C) with full-length PrP(Sc) and between copper-treated full-length PrP(C) with full-length PrP(Sc), antibody binding to residues 143-155 and 175-185 was consistently increased on PrP(Sc). Collectively, our results suggest that copper-treated full-length PrP(C) does not resemble full-length PrP(Sc), despite acquiring PrP(Sc)-like properties. In addition, since each full-length protein reacts distinctively to some of the antibodies, this binding pattern could discriminate between PrP(C) and PrP(Sc).     

An improved immunohistostaining procedure for peptides in human brain

Floating sections from human brains immersed for more than forty years in formalin, or from brains freshly fixed for a short time are treated by KMnO4-Pal's modified solutions to suppress the endogenous peroxidase activity before using the peroxidase-antiperoxidase method (PAP), or to remove the autofluorescence of lipofuscin, which is very intense in brains from old patients, before using the immunofluorescence method. Following this, immersion of sections in NaOH and H2O2 allows for the demasking of antigenic sites. These treatments enhance the immunolabelling considerably, with results comparable to those obtained with freshly fixed tissues, and facilitate the discrimination between specifically and unspecifically stained structures.     

Sex-dependent action of prenatal fractionated X-irradiation in the mouse. Biochemical findings.

X-irradiation of pregnant NMRI-mice on gestational days 11-13 with 3 x 10.5 Gy increased postnatal mortality of the female offspring only. Weights, protein content and acetylcholinesterase, as well as Na,K-ATPase activities in the brains of all treated offspring, were changed. There were, however, no differences between females and males with respect to these parameters.     

Nonhistone protein with high affinity for histone H1 and HMG 14 protein

Summary Specific interaction of a nonhistone protein from mouse spleen chromatin with histones HI, H2A and HMG 14 protein is shown. Some implications of these findings are briefly discussed.     

Tissue survey of mammalian modulator-dependent protein kinases

Summary The levels of modulator-dependent protein kinases and protamine-dependent protein kinase(s) in various tissues of adult mice were compared. Cerebellum contained the highest levels of both modulator-dependent protein kinases and protamine-dependent protein kinase(s), whereas skeletal muscle contained no detectable enzymes. The lung and the ileum were also rich in modulator-dependent protein kinases, while other tissues were poor sources of these enzymes.Acknowledgments. This work was supported by grants (RR-08119-PK project from the National Institutes of Health, USA. and CDP-8004200 from the National Science Foundation, USA.     

Separation between modulator-dependent protein kinases I and II

The separation of modulator-dependent protein kinase I from modulator-dependent protein kinase II obtained from the lungs of sexually premature male mice was accomplished by Sephadex G-200 gel filtration. After preincubation of a mouse lung cytosol fraction with arginine-rich histone, theophylline, cyclic GMP and crude protein kinase modulator a cyclic GMP-dependent protein kinase activity peak present in a non-preincubated sample completely disappeared and was replaced by a late-eluted modulator-dependent protein kinase II peak. There was a difference in substrate specificity between modulator-dependent protein kinase I and modulator-dependent protein kinase II despite their similar dependence on crude protein kinase modulator or partially purified stimulatory protein kinase modulator for their maximal activities.     

Expressed protein ligation: a resourceful tool to study protein structure and function

This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review, we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function.     

Myelin basic protein: a multifunctional protein

Myelin basic protein (MBP), the second most abundant protein in central nervous system myelin, is responsible for adhesion of the cytosolic surfaces of multilayered compact myelin. A member of the ‘intrinsically disordered’ or conformationally adaptable protein family, it also appears to have several other functions. It can interact with a number of polyanionic proteins including actin, tubulin, Ca²⁺-calmodulin, and clathrin, and negatively charged lipids, and acquires structure on binding to them. It may act as a membrane actin-binding protein, which might allow it to participate in transmission of extracellular signals to the cytoskeleton in oligodendrocytes and tight junctions in myelin. Some size isoforms of MBP are transported into the nucleus and thus they may also bind polynucleotides. Extracellular signals received by myelin or cultured oligodendrocytes cause changes in phosphorylation of MBP, suggesting that MBP is also involved in signaling. Further study of this very abundant protein will reveal how it is utilized by the oligodendrocyte and myelin for different purposes. Received 2 March 2006; received after revision 12 April 2006; accepted 16 May 2006     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Sex-dependent action of prenatal fractionated X-irradiation in the mouse. Biochemical findings

Summary X-Irradiation of pregnant NMRI-mice on gestational days 11–13 with 3×1.05 Gy increased postnatal mortality of the female offspring only. Weights, protein content and acetylcholinesterase, as well as Na, K-ATPase activities in the brains of all treated offspring, were changed. There were, however, no differences between females and males with respect to these parameters.We gratefully acknowledge the assistance of Miss C. Gutmann.     

Purification of acidic Z protein from human liver

Summary Preparation of Z protein from human liver is described. Z protein consists of 2 forms which have different isoelectric points, pI 5.8 and pI 8.7, respectively. The acidic Z protein has a molecular weight of about 11,000 and has binding affinity for BSP using gel filtration.     

Cellular maintenance of nuclear protein homeostasis

The accumulation and aggregation of misfolded proteins is the primary hallmark for more than 45 human degenerative diseases. These devastating disorders include Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. Over 15 degenerative diseases are associated with the aggregation of misfolded proteins specifically in the nucleus of cells. However, how the cell safeguards the nucleus from misfolded proteins is not entirely clear. In this review, we discuss what is currently known about the cellular mechanisms that maintain protein homeostasis in the nucleus and protect the nucleus from misfolded protein accumulation and aggregation. In particular, we focus on the chaperones found to localize to the nucleus during stress, the ubiquitin–proteasome components enriched in the nucleus, the signaling systems that might be present in the nucleus to coordinate folding and degradation, and the sites of misfolded protein deposition associated with the nucleus.     

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.     

Mechanisms of protein toxicity in neurodegenerative diseases

Protein toxicity can be defined as all the pathological changes that ensue from accumulation, mis-localization, and/or multimerization of disease-specific proteins. Most neurodegenerative diseases manifest protein toxicity as one of their key pathogenic mechanisms, the details of which remain unclear. By systematically deconstructing the nature of toxic proteins, we aim to elucidate and illuminate some of the key mechanisms of protein toxicity from which therapeutic insights may be drawn. In this review, we focus specifically on protein toxicity from the point of view of various cellular compartments such as the nucleus and the mitochondria. We also discuss the cell-to-cell propagation of toxic disease proteins that complicates the mechanistic understanding of the disease progression as well as the spatiotemporal point at which to therapeutically intervene. Finally, we discuss selective neuronal vulnerability, which still remains largely enigmatic.     

Coronavirus envelope protein: A small membrane protein with multiple functions

Coronavirus envelope protein is a small membrane protein and minor component of the virus particles. It plays important roles in virion assembly and morphogenesis, alteration of the membrane permeability of host cells and virus-host cell interaction. Here we review recent progress in characterization of the biochemical properties, membrane topology and functions of the protein. Received 27 February 2007; received after revision 4 April 2007; accepted 26 April 2007