Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.     

Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.     

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms,and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.Rotaviruses are a major cause of acute gastroenteritis in infants and young children, producing a high burden of disease worldwide and over 600,000 deaths per annum, mainly in developing countries (43). Recently, two live attenuated rotavirus vaccines (49, 58) have been licensed in various countries, and their widespread use in universal mass vaccination programs is being implemented (55).Rotaviruses form a genus of the Reoviridae family. They contain a genome of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). After entry into the host cell the outer layer of the triple-layered particles (TLPs; infectious virions) is removed in endocytic vesicles, and the resulting double-layered particles (DLPs) actively transcribe mRNAs from the 11 RNA segments and release them into the cytoplasm. The mRNAs are translated into proteins but also act as templates for dsRNA synthesis (RNA replication). The early stages of viral morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed “viroplasms.” Partially assembled DLPs are released from viroplasms and receive their outer layer in the rough endoplasmic reticulum (RER), forming TLPs (for details, see Estes and Kapikian [20]).The rotavirus nonstructural proteins NSP2 and NSP5 are major components of viroplasms (20, 47). These two proteins alone are sufficient to induce the formation of viroplasm-like structures (VLS) (21). Blocking of either NSP2 or NSP5 in rotavirus-infected cells significantly reduces viroplasm formation and the production of infectious viral progeny (11, 54, 57). Until now, host cell proteins involved in viroplasm formation have not been identified.Morphological similarities between viroplasms and lipid droplets (LDs) prompted us to investigate their relationship. Both structures have phosphoproteins (NSP5 and perilipin A, respectively) inserted on their surface in ringlike shapes (16, 34). LDs are intracellular organelles involved in lipid and carbohydrate metabolism. They consist of a neutral lipid core surrounded by a phospholipid monolayer containing LD-associated proteins; those include proteins of the PAT family consisting of perilipin, adipophilin (adipose differentiation-related protein [ADRP]), and TIP-47 (9, 37). Lipolysis from LDs is regulated by hormones such as catecholamines, which trigger the phosphorylation of hormone-sensitive lipase (HSL) and perilipin A and induce LD fragmentation. Incubating adipocytes with the β-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) activates this pathway (27, 34). LD formation can also be blocked by triacsin C, a specific inhibitor of long chain acyl coenzyme A synthetases (30, 39).We demonstrate here that rotavirus viroplasms colocalize with the LD-associated proteins perilipin A and ADRP and also with the lipids of LDs. These interactions appear to be required for the formation of functional viroplasms and the production of infectious viral progeny, since compounds dispersing LDs or blocking LD formation significantly decrease the number and size of viroplasms and the amount of infectious progeny. Taken together, these findings strongly suggest a critical role of LDs in rotavirus replication.     

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of α-Synuclein Aggregation

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein α-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.     

ABC A-subfamily transporters: structure, function and disease

ABC transporters constitute a family of evolutionarily highly conserved multispan proteins that mediate the translocation of defined substrates across membrane barriers. Evidence has accumulated during the past years to suggest that a subgroup of 12 structurally related "full-size" transporters, referred to as ABC A-subfamily transporters, mediates the transport of a variety of physiologic lipid compounds. The emerging importance of ABC A-transporters in human disease is reflected by the fact that as yet four members of this protein family (ABCA1, ABCA3, ABCR/ABCA4, ABCA12) have been causatively linked to completely unrelated groups of monogenetic disorders including familial high-density lipoprotein (HDL) deficiency, neonatal surfactant deficiency, degenerative retinopathies and congenital keratinization disorders. Although the biological function of the remaining 8 ABC A-transporters currently awaits clarification, they represent promising candidate genes for a presumably equally heterogenous group of Mendelian diseases associated with perturbed cellular lipid transport. This review summarizes our current knowledge on the role of ABC A-subfamily transporters in physiology and disease and explores clinical entities which may be potentially associated with dysfunctional members of this gene subfamily.