This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and ...useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Phone: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.
The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in ...organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected ≈ 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Δ mutation increases the petite-inducing phenotype of an abf2Δ mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of ρ+mtDNA.
The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. ...Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.
Disabled 1 (Dab1) functions as a critical adapter protein in the Reelin signaling pathway to direct proper positioning of neurons during brain development. Reelin stimulates phosphorylation of Dab1 ...on tyrosines 198 and 220, and phosphorylated Dab1 is likely to interact with downstream signaling proteins that contain Src homology 2 (SH2) domains. To search for such proteins, we used a Sepharose-conjugated peptide containing phosphotyrosine 220 (PTyr-220) of Dab1, as an affinity matrix to capture binding proteins from mouse brain extracts. Mass spectrometric analysis of bound proteins revealed that Crk family adapter proteins selectively associated with this phosphorylation site. We further show that Crk-I and Crk-II, but not CrkL, stimulate phosphorylation of Dab1 on tyrosine 220 in a Src-dependent manner. Our results suggest that Crk family adapter proteins may play an important role in the Reelin signaling pathway during brain development.
The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated ...glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after ...cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.
The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 ...CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.
The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this ...activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.
The PITSLRE protein kinases, hereafter referred to as cyclin-dependent kinase 11 (CDK11) due to their association with cyclin
L, are part of large molecular weight protein complexes that contain RNA ...polymerase II (RNAP II) as well as numerous transcription
and RNA processing factors. Data presented here demonstrate that the influence of CDK11 p110 on transcription and splicing does not involve phosphorylation of the RNAP II carboxyl-terminal domain by CDK11 p110 . We have isolated a DRB- and heparin-sensitive protein kinase activity that co-purifies with CDK11 p110 after ion exchange and affinity purification chromatography. This protein kinase was identified as casein kinase 2 (CK2)
by immunoblot and mass spectrometry analyses. In addition to the RNAP II carboxyl-terminal domain, CK2 phosphorylates the
CDK11 p110 amino-terminal domain. These data suggest that CDK11 p110 isoforms participate in signaling pathways that include CK2 and that its function may help to coordinate the regulation of
RNA transcription and processing events. Future experiments will determine how phosphorylation of CDK11 p110 by CK2 specifically affects RNA transcription and/or processing events.