Anti-Mup44 autoantibodies, which are targeted to cN1A, represent the first serological biomarker for sIBM and may facilitate the diagnosis of this type of myositis.

Despite years of investigation, the molecular mechanism responsible for regulation of the c-Abl tyrosine kinase has remained elusive. We now report inhibition of the catalytic activity of purified c-Abl in vitro, demonstrating that regulation is an intrinsic property of the molecule. We show that the interaction of the N-terminal 80 residues with the rest of the protein mediates autoregulation. This N-terminal "cap" is required to achieve and maintain inhibition, and its loss turns c-Abl into an oncogenic protein and contributes to deregulation of BCR-Abl.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.energy metabolism | phototransduction A significant energy distribution problem can arise from the relative locations of mitochondria, ion pumps, and synapses in neurons. In photoreceptors, ion pumps occupy the intervening space between the centrally located mitochondria and the synaptic terminal. Ion pumping in dark-adapted photoreceptors consumes ∼20× more energy than neurotransmission (1). Therefore, the pumps could intercept all the metabolic energy made by the mitochondria before it can reach the synaptic terminal. In the vascularized retinas of mice, rats, and humans (2-4) this problem is solved by the presence of additional mitochondria in the terminal. However, in the avascular retinas of zebrafish, salamanders, rabbits, and guinea pigs there are no mitochondria in the terminals (2, 4, 5), which creates a need to partition some of the energy made by the central mitochondria into a protected form that can bypass the ion pumps to support the essential energy demands of the synaptic terminal.Energy consumption within retinal photoreceptors is compartmentalized and light-dependent. During illumination, phototransduction and light adaptation consume energy in the outer segment (OS). In darkness, energy is consumed by ion pumps in the inner segment and by glutamate release at the synaptic terminal (1). Energy demands and O 2 consumption are far greater in darkness than in light (1, 6-8).Metabolic energy is distributed in most cells as either ATP or phosphocreatine (PCr). There are 2 isoforms of creatine kinase (CK) in neurons, ubiquitous mitochondrial creatine kinase (uMtCK), and brain-type cytoplasmic creatine kinase (CK-B). uMtCK creates PCr from ATP at mitochondria (9), and CK-B can recreate ATP from PCr at sites of energy demand. In this way uMtCK and CK-B can collaborate to transfer metabolic energy between neuronal compartments (10, 11). This paper descr...

The eukaryotic endonucleases RNase P and RNase MRP require both RNA and protein subunits for function. Even though the human RNase P and MRP RNAs were previously characterized, the protein composition of the particles remains unknown. We have identified a human a Caenorhabditis elegans sequence showing homology to yPop1, a protein subunit of the yeast RNase P and MRP particles. A cDNA containing the complete coding sequence for the human protein, hPop1, was cloned. Sequence analysis identifies three novel sequence motifs, conserved between the human, C. elegans and yeast proteins. Affinity‐purified anti‐hPop1 antibodies recognize a single 115 kDa protein in HeLa cell nuclear extracts. Immunoprecipitations with different anti‐hPop1 antibodies demonstrate an association of hPop1 with the vast majority of the RNase P and MRP RNAs in HeLa cell nuclear extracts. Additionally, anti‐hPop1 immunoprecipitates possess RNase P enzymatic activity. These results establish hPop1 as the first identified RNase P and MRP protein subunit from humans. Anti‐hPop1 antibodies generate a strong nucleolar and a weaker homogeneous nuclear staining in HeLa cells. A certain class of autoimmune patient serum precipitates in vitro‐translated hPop1. hPop1 is therefore an autoantigen in patients suffering from connective tissue diseases.

No abstract

The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 59 end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20-and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus-(VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.

Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the various steps of ribosome synthesis (reviewed in references 30 and 46). Several snoRNAs have previously been shown to be required for pre-rRNA processing (12,30,46), and a large set of snoRNAs is involved in ribose methylation and pseudouridylation of rRNA (8,15,24,33,34,49). snoRNAs are heterogeneous in size, structural elements, and protein association. They are produced by two biosynthetic pathways. Most snoRNAs are encoded within the pre-mRNA introns of ribosomal or nucleolar proteins. The processing of such premRNAs via endo-and exonucleolytic cleavages results in the generation of mature noncapped snoRNAs (30). Other snoRNAs, e.g., U3 snoRNA, are transcribed from independent genes and typically possess a modified 5Ј terminus, usually a 5Ј trimethylguanosine (TMG) cap (30).snoRNPs can be divided into four groups, which appear to be functionally distinct (1, 46). Methylation guide snoRNPs direct the site-specific formation of 2Ј-O-methyl groups in mature rRNA. All snoRNAs of this class contain two conserved sequence elements, referred to as box C and box D (30), and contain an extended region (10 to 21 nucleotides) of base complementarity to mature rRNA (8,24,34,49). Members of the second group of snoRNAs, which encompasses U3, U8, U14, and U22 snoRNAs, also contain the conserved box C and D elements and are involved in pre-rRNA processing reactions (reference 46 and references therein). All box C-and D-containing snoRNAs, including methylation guide snoRNAs, are associated with the conserved nucleolar protein fibrillarin, which thus is a common snoRNP component (30). Members of the third class of snoRNAs lack the box C and D elements but share another conserved sequence element, referred to as the ACA box (1). Such snoRNA...