Cloning and characterization of the

The combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of protein complexes and large protein-interaction networks. Emerging AP–MS approaches promise a better understanding of protein-complex stoichiometry, structural organization and the dynamics of protein-complex composition. The versatile combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP–MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP–MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein–complex assembly.

http://bio.ijs.si/~krizaj/group/Struktura%20proteinov%202012/Seminarji-osnovna%20literatura/4.Gingras2007.pdf

Analysis of protein complexes using mass spectrometry

Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Kinetoplastids are a group of flagellated protozoans that include the species Trypanosoma and Leishmania, which are human pathogens with devastating health and economic effects. The sequencing of the genomes of some of these species has highlighted their genetic relatedness and underlined differences in the diseases that they cause. As we discuss in this Review, steady progress using a combination of molecular, genetic, immunologic, and clinical approaches has substantially increased understanding of these pathogens and important aspects of the diseases that they cause. Consequently, the paths for developing additional measures to control these "neglected diseases" are becoming increasingly clear, and we believe that the opportunities for developing the drugs, diagnostics, vaccines, and other tools necessary to expand the armamentarium to combat these diseases have never been better.

/pdf/kinetoplastids-related-protozoan-pathogens-different-h01mck3txj.pdf

Kinetoplastids: related protozoan pathogens, different diseases

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.

/pdf/dead-box-proteins-a-family-affair-active-and-passive-players-fx8i3yme6c.pdf

Dead-box proteins: a family affair—active and passive players in RNP-remodeling

http://www.bio.umass.edu/micro/klingbeil/590s/Reading/Stuart2005.pdf

Complex management: RNA editing in trypanosomes

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed.

/pdf/identification-of-novel-components-of-trypanosoma-brucei-3s0f7hea9m.pdf

Identification of novel components of Trypanosoma brucei editosomes.

RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and ≈20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).

https://www.pnas.org/content/pnas/102/46/16614.full.pdf

An essential RNase III insertion editing endonuclease in Trypanosoma brucei.

https://schnauferlab.bio.ed.ac.uk/PDFs/Schnaufer%20et%20al%20MolCell%202003.pdf

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3*-terminal uridylyl transferase, 3*-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an ;49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

/pdf/association-of-two-novel-proteins-tbmp52-and-tbmp48-with-the-2lw8tj95w8.pdf

Nancy Lewis Ernst

Papers

Complex management: RNA editing in trypanosomes

Identification of novel components of Trypanosoma brucei editosomes.

An essential RNase III insertion editing endonuclease in Trypanosoma brucei.

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.

Association of Two Novel Proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex