Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei

Huang, Lan; Shen, Min; Chernushevich, Igor V.; Burlingame, Alma L.; Wang, Ching C.; Robertson, Colin D.

doi:10.1016/s0166-6851(99)00096-1

Cited by 31 publications

(33 citation statements)

References 23 publications

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“…All of the catalytic subunits share considerable sequence identities from T. brucei to human with complete conservation of all pivotal active site residues (39). Most likely, additional structural requirements on the rest of protein backbone in T. brucei 20 S proteasome also play essential roles (40).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Wang

Bozdech

Liu

et al. 2003

Journal of Biological Chemistry

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We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven ␣-and seven ␤-subunits. Of the seven ␤-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three ␤-subunits were labeled by the affinity reagents, only two ␤-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to ␤2 and ␤5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal ␤1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.

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Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Wang

Bozdech

Liu

et al. 2003

Journal of Biological Chemistry

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“…Postacquisition, data are automatically base line-corrected and smoothed, and peaks are stored in an Oracle TM data base. Peak lists from all MS and MSMS spectra were automatically extracted out of the Oracle TM data base and submitted for batch MS-Fit and batch MS-Tag data base searching (prospector.ucsf.edu) (9).…”

Section: Fig 2-continuedmentioning

confidence: 99%

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Allen

Patel

Huang

et al. 2002

Molecular & Cellular Proteomics

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The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA.More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K D ‫؍‬ 1.5 M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

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“…Preparation and electrophoretic separation of 20 S proteasomes from T. brucei was performed as described previously (25,29). After silver staining, protein Spots were excised from the gel.…”

Section: Protein Separation By Two-dimensional Gel Electrophoresis Anmentioning

confidence: 99%

“…A static data base, non-redundant Static, was generated by downloading the non-redundant National Center for Biotechnology Information protein data base (as of 3/11/99) and modified by removing sequences matching 3 of our query sets that had been submitted to the data base following their identification early in this project (29). All searches used either the BLOSUM62MS scoring matrix, a version of BLOSUM62 (36), modified to account for Ile/Leu and Gln/Lys ambiguities associated with determination of protein sequences using mass spectrometry (37) or the PAM30MS matrix, modified in an analogous manner from the PAM30 matrix (38).…”

Section: Data Base Searching and Analysis Of Sequence Informationmentioning

confidence: 99%

“…These were aligned with the individual peptide sequences using either Pileup (41) or ClustalW (42). To evaluate the correctness of the linear ordering, we subsequently compared the ordering of each catenated sequence to the ordering of those same peptides within the sequences of the fulllength proteasome protein subunits that were obtained independently from DNA sequencing after the completion of this study (29). 4 While the data base search scores for these full-length sequences were useful for comparison with scores obtained for sequence fragments obtained from sequencing by mass spectrometry, these full-length sequences were not used in the bioinformatic analysis of the sequences obtained from mass spectrometry.…”

Section: Data Base Searching and Analysis Of Sequence Informationmentioning

confidence: 99%

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Functional Assignment of the 20 S Proteasome from Trypanosoma brucei Using Mass Spectrometry and New Bioinformatics Approaches

Huang¹,

Jacob²,

Pegg³

et al. 2001

Journal of Biological Chemistry

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As experimental technologies for characterization of proteomes emerge, bioinformatic analysis of the data becomes essential. Separation and identification technologies currently based on two-dimensional gels/mass spectrometry provide the inherent analytical power required. This strategy involves protein spot digestion and accurate mass mapping together with computational interrogation of available data bases for protein functional identification. When either no exact match is found or when the possible matches only partially account for molecular weights actually observed, peptide sequencing by tandem mass spectrometry has emerged as the methodology of choice to provide the basic additional information required. To evaluate the capabilities of bioinformatics methods employed for identifying homologs of a protein of interest, we attempted to identify the major proteins from the 20 S proteasome of Trypanosoma brucei using sequence information determined using mass spectrometry. The results suggest that neither the traditional query engines, BLAST and FASTA, nor specialized software developed for analysis of sequence information obtained by mass spectrometry are able to identify even closely related sequences at statistically significant scores. To address this deficit, new bioinformatics approaches were developed for concomitant use of the multiple fragments of short sequence typically available from methods of tandem mass spectrometry. These approaches rely on the occurrence of congruence across searches of multiple fragments from a single protein. This method resulted in sharply better statistical significance values for correct hits in the data base output relative to that achieved for independent searches using single sequence fragments.Fueled by the genome projects, encyclopedic increases in the banking of newly obtained, comprehensive biological data are transforming studies of biology and medicine (1). As the postgenomic era moves into high gear, new "high throughput" technologies are allowing characterization of gene expression profiles, comparisons of genomic complements, and identification of the genetic markers associated with normal, pathological, or environmentally triggered states. Yet information derived from full analysis of genomics alone is clearly inadequate to explain the complexities of cell biology. Recent studies showing differences between the genome and the proteome suggest that the profound understanding we seek will require the complete and direct characterization of the proteome as well (2, 3).Peptide mass mapping by MALDI-TOF 1 MS (4) or liquid chromatography-electrospray ionization MS (5, 6), combined with interrogation of sequence data bases (7-12), currently is the most widely employed strategy for the identification of expressed proteins. This methodology involves electrophoretic separation of proteins at sub-picomole levels, digestion with trypsin, and measurement of the molecular weights of the resulting peptide mixture by mass spectrometry. This strategy can routinely identify p...

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Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei

Cited by 31 publications

References 23 publications

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Functional Assignment of the 20 S Proteasome from Trypanosoma brucei Using Mass Spectrometry and New Bioinformatics Approaches

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