116
IntroductionLarge-scale proteomic profiling of biological samples such as cells, organelles or biological fluids has led to discovery of numerous key and novel proteins involved in many biological/disease processes, as well as to the identification of novel disease biomarkers and potential therapeutic targets. Bioinformatics infrastructure and systems play instrumental roles in such data analyses and discovery processes. iProXpress (integrated Protein eXpression system) is an prototype analysis system designed to help analyze proteomic and genomic data, such as protein/peptide and gene profiles from MS proteomic and microarray gene expression experiments. It has been applied to several studies including the expression profile analysis of hormone-induced changes in tumor cells, and is currently being adopted for analyses of pathogen/host genomic and proteomic data produced from the NIAID Biodefense Proteomic Program. We will present a case study where proteomes of various stages of melanosomes from human melanoma cells were analyzed using the iProXpress system to illustrate its utility in facilitating a better understanding of pathways of melanin synthesis and melanosome biogenesis.
The iProXpress Knowledge System for Proteomic Data AnalysisZhang-Zhi Hu1; Hongzhan Huang1; Peter McGarvey1; An Chi2; Julio Valencia3; Cathy H. Wu1
1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20007; 2 University of Virginia Department of Chemistry, Charlottesville, VA 22904; 3Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892
Protein Information
Matrix
iProXpress
integrated Protein eXpression Analysis System
Functional Annotation
Expression Profiling
Protein Mapping
IP/2D/MS Proteomic DataGene Expression
Gene/Protein ID list Peptide Sequence
UniProt
iProClass
Function Categorization Chart
Two-Way Comparison Matrix
Pathway MapInteraction Map
GO tree visualization
Protein Information Matrix
Figure 1. An Overview of the iProXpress System
System Designs• iProXpress consists of three major components:
• The PIR (Protein Information Resource) data warehouse with integrated protein information, • Analytical tools for sequence analysis and functional annotation, and • Graphical user interface for categorization and visualization of expression data.
• Major Functionalities• Gene/Peptide to Protein Mapping. Gene or protein lists are mapped to corresponding entries in UniProt
Knowledgebase (UniProtKB) of all known proteins based on gene/protein IDs, names or sequences. • Protein information matrix. A comprehensive matrix is generated, summarizing salient features including gene
ontology (GO) and pathways retrieved from underlying PIR protein databases with annotated experimental literature information or inferred based on sequence similarity.
• Protein Data Analysis. By iterative categorization and sorting of proteins in the information matrix, users can correlate expression/interaction patterns to protein properties for pathway and network discovery.
Melanosome Proteome Analysis• ID mapping. Total 2298 gi numbers were mapped using UniProt/PIR ID mapping service, which converts common
gene/protein IDs (e.g., gi number) to UniProtKB AC/ID and vice versa. 1253 were mapped to UniProtKB sequences. • Peptide mapping. Peptides from MS data were matched against all human sequences in UniProtKB with a two-step
procedure: direct mapping or mapping using UniRef90 clusters (90% or more sequence identity grouped in the cluster), giving 1506 mapped proteins. Combining both ID and peptide mappings, 1936 (84%) of the proteins were mapped to 1532 UniProtKB sequences.
• Protein information matrix. A comprehensive protein information matrix were generated from underlying PIR data warehouse (UniProt/iProClass) or inferred based on sequence homologies for melanosome proteins. Attributes in the matrix include protein name, family, domain/motif/site, isoform, post-translational modification, GO, function/functional category, structure/structural classification, pathway, protein interaction and complex, etc.
• Melanosome proteome analysis: The melanosome proteome datasets were partitioned into 12 groups and categorization and sorting functions for each group or all datasets were provided based on the protein information matrices especially of gene ontologies (GO) and pathways (KEGG and BioCarta). Iterative categorization and sorting of proteins based on functions, pathways, and/or other attributes were carried out to generate various protein clusters, from which interesting unique or common proteins at different stages of melanosome biogenesis can be identified in combination with manual examination.
• Comparative analysis of organelle proteomes. Melanosome proteomes of early or late stages were compared with other organelle proteomes such as lysosome, synaptosome and endosome. This comparative analysis coupled with other bioinformatic analysis was aimed at deducing a set of signature proteins characteristics of the melanosme.
Conclusion
The prototype iProXpress system enables the analysis of large-scale expression data through its functionalities in ID/peptide mapping, sequence analysis, functional annotation, and functional profiling. The case study on melanosome proteome analysis illustrates that the use of the system greatly facilitates the study of melanosome biogenesis and pigmentation related human diseases. The system should be applicable to a broad range of genomic and proteomic profiling studies.
Sorting of data sets and display of protein information matrix
Functional categorization based on gene ontology
Two-way comparative matrix
NIAID Biodefense Proteomics Program Mater catalog of proteins from NIAID Biodefence Proteomic Research Centers.
A catalog of proteins from Harvard University, cDNA clones of which are used for protein chip assays to detect protein-protein interactions in situ.
Browsing selected complete proteome(s) with protein links to the Proteomic Center data.
All stage IV-specific membrane protein: P50443 : Sulfate transporter (Solute carrier family 26 member 2) (OMIM: 600972)Q9NZ45 : Protein C10orf70 P33121 : Long-chain-fatty-acid--CoA ligase 1 (LACS 1) (OMIM: 152425) (also in ER)Q8NCC2 : Hypothetical protein FLJ90355 (Solute carrier family 2Q8IWB8 : CCR4-NOT transcription complex, subunit 1, isoform bP27449 : Vacuolar ATP synthase 16 kDa proteolipid subunit (EC 3.6.3.14) (OMIM: 108745)Q71RS6 : Ion transporter JSX [Homo sapiens] – human skin color geneQ6ZTT7 : Hypothetical protein FLJ44232 [Homo sapiens]Q16444 : Phosphoglycerate kinase (Fragment) [Homo sapiens] (47 aa)
Table 1. Mapping of mouse color genes to human melanosome proteins Table 2. Partial list of stage-specific melanosome proteins
Figure 2. Electron microscopy images showing the morphological characteristics of the melanosomes during maturation. In pigmented cells such as MNT1, stage I melanosomes are relatively spherical organelles with an amorphous matrix; at Stage II, organelles elongate, the internal fibrils form regular parallel arrays and melanin synthesis starts at granular sites studding the internal walls of the melanosome; at late-stage stage III, deposition of melanin on the melanosomal matrix becomes evident; till stage IV, the organelle is fully matured with a heavy accumulation of melanin. For those of non-pigmented mutant cells such as SkMel-28, melanosomes stay at early developmental stages and melanin synthesis is halted due to the dysfunctional maturation and a large number of early stage melanosomes are accumulated as demonstrated on the panels to the right.
MNT1 Stage I (77)
P36955 : Pigment epithelium-derived factor precursor (PEDF) Q14254 : Flotillin-2 (Epidermal surface antigen)P07093 : Glia derived nexin precursor (GDN)P24390 : ER lumen protein retaining receptor 1 (KDEL receptor 1) (TM)O14880 : Microsomal glutathione S-transferase 3 (TM)
MNT1 Stage II (112)
Q12846 : Syntaxin-4 (TM) (interact with O75379 Vesicle-associated membrane protein 4; Q15836 Vesicle-associated membrane protein 3)Q04656 : Copper-transporting ATPase 1 (=mouse Atp7a)Adaptor proteins:O95782: AP-2 (~mouse Ap3bl) (also Skmel)Q96EL6: Adaptin (~mouse Ap3d)Vacuolar protein sorting:Q96A65 : Exocyst complex component Sec8P46459 : Vesicle-fusing ATPase (EC 3.6.4.6) (interact)P14415 : Sodium/potassium-transporting ATPase beta-2 chain (TM)
MNT4 Stage IV (267)
Adaptor protein: Q9Y6Q5 : Adaptor protein complex AP-1 mu-2 subunit, (interact with P63010: AP2B1) Motor poteins:Q14203 : Dynactin-1 (Progressive lower motor neuron disease [OMIM:607641]) (interact with P18669: Phosphoglycerate mutase 1)Q9H193 : KINESIN-13A2Transport:Q99747 : Gamma-soluble NSF attachment protein (also Skmel)Q99698 : Lysosomal trafficking regulator (=mouse Lyst)Vacuolar protein sorting:Q9H444 : VPS32 (~mouse Vps33a) (interact) (also Skmel)Q9NZZ3 : VPS60 (~mouse Vps33a)
Skmel28 unique (143)
Both melanosome-specific proteins Tyrosinase and TYRP1 are absent in Skmel28 data set, suggesting that their absence can partially account for the lack of melanin production in Skmel28. P57729 : Ras-related protein Rab-38 (=mouse Rab38)P51810 : G-protein coupled receptor 143 (=mouse Gpr143) P53794 : Sodium/myo-inositol cotransporter (Na(+)/myo-inositol cotransporter)
Slominski, A. et al. Physiol. Rev. 84: 1155-1228 2004; Copyright ©2004 American Physiological Society
Figure 3B. Schematic of melanosomal proteins (Hypothetical depiction)
Gene Symbol
Murine LocusFunction in
PigmentationHuman Melanosome
ProteinsHuman Disease (OMIM)
Ednrb piebald spotting (s)melanoblast differentiation
P24530 : ET-B (Common stage1& MNT1 stage2)
Waardenburg-shah syndrome [277580]…
Sfxn1 flexed tailTricarboxylate carrier
Q9H9B4 : Sideroflexin-1(Common early stage)
*Rab38 chocolate (cht)Targeting of Tyrp1 protein to the melanosome
P57729 : Ras-related protein Rab-38. (uniqueSkmel28)
606281
*si silver (si)melanosomal matrix protein
P40967 : Pmel 17 precursor (Common all stage)
Oculocutaneous albinism [155550]
*Tyr albino, color (c)melanogenic enzyme
P14679 : Tyrosinase precursor (Unique MNT1)
OCA1 [203100]; OCA1B [606952]…
*Tyrp1 brown (b)melanosomal enzyme/stabilizing factor
P17643 : 5,6-dihydroxyindole-2-carboxylic acid oxidase precursor. (Unique MNT1)
Rufous albinism, ROCA [115501]; OCA3 [203290];Precocious graying of hair [278400]
*Gpnmbiris pigment dispersion (ipd)
Apparent melanosomal component
Q14956 : Transmembrane glycoprotein NMB precursor (Common all stage)
Glaucoma-related pigment dispersion syndrome-1 [604368 ]
*Matp underwhite (uw) transporter
Q9UMX9 : Melanoma antigen AIM1 Q6P2P0 : Membrane-associated transporter protein, isoform b (MNT1 stage1 & 2)
OCA4 [606574]
Rab27a ashen (ash)melanosome transport
Q6IAS8 : RAB27A proteinP51159 : Ras-related protein Rab-27A (Common all stage)
Griscelli syndrome, type 2 [607624]
gdn golden (gdn)
Causes delayed and reduced development of melanin pigmentation
Q71RS6Ion transporter JSX (Unique late stage)
SLC24A5, a human skin color geneScience 16 December 2005: 1754-1755.
This is a partial list of total 17 mapped genes. The others include Lyst, Ostm1, Dct, Atp7a, Gpr143, Myo5a and Krt2-17. For complete list, go to http://pir.georgetown.edu/~huz/datamining/proteomics/
Organelle # Protein reported
# Entries mapped
Common with melanosome *
References
Mouse ER (ER) ~141 131 57 (M:51, S:36)-19(33%) all stage
Knoblach et al, 2003
Human neuromelanin granules (NG)
72 72 43 (M:38, S:36)-22(51%) all stages
Tribl et al, 2005
Rat synaptosome (SY)
200 88 43 (M:35, S:27)-14(33%) all stages
Witzmann et al, 2005
Rat lysosome (LY) 215 116 49 (M:40, S:38)-13(27%) all stages
Bagshaw et al, 2005
Human platelet (PL) ~93 71 33 (M:26, S:22)-6(18%) all stages
Martens et al, 2005
Human exosome (EX)
~56 55 42 (M:42, S:32)-18(43%) all stages
Mears et al, 2004Proteins Present in at Least 3 Other Organelles in Either Early Stages or All Stages of
Melanosomes (1-ER, 2-NG, 3-SY, 4-EX, 5-PL, 6-LY)
1 2 3 4 5 6P30101 |+|+|+|+|+||+|+|-|+|-|-| Protein disulfide-isomerase A3 precursor P35232 |+|+|-|-|+||+|-|+|+|-|+| Prohibitin P60709 |+|+|+|-|-||+|-|+|+|-|-| Actin, cytoplasmic 1 (Beta-actin)Q5FVE7 |+|+|-|-|-||+|-|+|-|-|+| Voltage-dependent anion channel 1P04406 |+|+|+|+|+||-|+|+|-|-|+| Glyceraldehyde-3-phosphate dehydrogenaseP04843 |+|+|+|+|+||-|+|-|-|+|+| protein glycosyltransferaseP06576 |+|+|+|-|+||+|+|+|-|-|-| ATP synthase beta chain, mitochondrialP10809 |+|+|+|+|+||-|+|+|+|-|-| 60 kDa heat shock protein, mitochondrialP11142 |+|+|+|+|+||-|+|+|+|-|+| Heat shock cognate 71 kDa protein
* M – MNT1 cells ; S – Skmel28 cells; Percent of protein common in all stages of MNT1 melanososmes are also indicated. The complete listing of the common proteins is at http://pir.georgetown.edu/~huz/datamining/proteomics/ .
Table 3. Summary of the comparison of organellar proteomes
ER
trans-Golgi Network
cis-Golgi
Lysosome
AP3
Stage I
Stage IV
Stage III
Stage II
AP1
cell membrane
AP2
Early Endosome
Late Endosome
AP3
AP3APxAP3
AP4
Tyrgp100 (10%)
Gp100gp100
Tyr
AP3gp100
Tyrosinase
gp100 (90%)
Tyrp1, Dct and MART1 ?
Figure 3A. Trafficking and sorting of proteins to melanosomes. Hearing 2005.
Proposal: a list of proteins characteristic of melanasome that may consist of:
• previously known melanosome-specific proteins;
• current short list of melanosome stage-specific proteins as proposed in table 2;
• some constituent proteins commonly found in several other organelles such as those in table 3.
A Case Study – Organellar proteomes of various stages of melanosomes from human melanoma cell lines• Mapping to known mouse coat color genes led to identification of 17 essential human melanosome related proteins; • Identification of possible stage-specific melanosome proteins for validation; • Comparison of melanosome proteome with those of several other organelles.
Top Related