Transcriptomics

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Genomology
Proteomics
Transcriptomics
Interactomics
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FUNGAL TRANSPORTOSOMICS
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Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology
(NC-IUBMB) In consultation with the
IUPAC-IUBMB Joint Commission on Biochemical
Nomenclature (JCBN) Enzyme Nomenclature http//
www.chem.qmul.ac.uk/iubmb/enzyme/
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Enzyme Nomenclature
EC 1 Oxidoreductases EC 1.1 Acting on the CH-OH
group of donors EC 1.1.1 With NAD or NADP as
acceptor EC 1.1.1.1 alcohol dehydrogenaseEC
1.1.1.2 alcohol dehydrogenase (NADP)EC 1.1.1.3
homoserine dehydrogenaseEC 1.1.1.4
(R,R)-butanediol dehydrogenase EC 1.1.1.37
malate dehydrogenase
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EC NOMENCLATURE

• ONLY BASED ON CATALYTIC REACTIONS OF SOLUBLE
  ENZYMES WHICH ARE EXPERIMENTALY DEMONSTRATED
• NO TRANSPORT REACTIONS
• NO SEQUENCE /PHYLOGENY INFORMATION

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Proton-transporting P-type ATPases TMHMM
membrane span and topology prediction
M.jannaschii
M.thermoautotrophicum
M.thermoautotrophicum
T. acidophilum
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Transporter Classification
TCDB Milton Saier (validated by IUBMB)
1.
A.
1.
1.
1
First (number)
Second (letter)
Third
Fourth
Fifth
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Mechanisms
1. Channels/Pores
2. Secondary Transporters
3. Primary Active Transporters
9. Incompletely Characterized Transport Systems
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Superfamilies/Families

• 2.A. uniporters, symporters, antiporters
• 2.A.1 The Major Facilitator Superfamily (MFS)
• 2.A.2 The Glycoside/Pentoside/Hexuronide (GPH)
  Cation Symporter Family
• 2.A.3 The Amino Acid-Polyamine-Organocation (APC)
  Superfamily
• 2.A.4 The Cation Diffusion Facilitator (CDF)
  Family
• 2.A.5 The Zinc (Zn2)-Iron (Fe2) Permease (ZIP)
  Family
• 2.A.6 The Resistance-Nodulation-Cell Division
  (RND) Superfamily
• 2.A.73 The Inorganic Carbon (HCO3-) Transporter
  (ICT) Family
• 2.A.74 The 4 TMS Multidrug Endosomal Transporter
  (MET) Family
• 2.A.75 The L-Lysine Exporter (LysE) Family
• 2.A.76 The Resistance to Homoserine/Threonine
  (RhtB) Family
• 2.A.77 The Cadmium Resistance (CadD) Family

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Subfamilies

• 2.A. uniporters, symporters, antiporters
• 2.A.1 Major Facilitator Superfamily (MFS)
• 2.A.1.1 The Sugar Porter (SP) Family
• 2.A.1.2 The DrugH Antiporter-1 (12 Spanner)
  (DHA1) Family
• 2.A.1.3 The DrugH Antiporter-2 (14 Spanner)
  (DHA2) Family
• 2.A.1.4 The OrganophosphatePi Antiporter (OPA)
  Family
• 2.A.1.5 The OligosaccharideH Symporter (OHS)
  Family
• 2.A.1.6 The MetaboliteH Symporter (MHS) Family
  
• 2.A.1.7 The FucoseH Symporter (FHS) Family
• 2.A.1.8 The Nitrate/Nitrite Porter (NNP) Family
• 2.A.1.9 The PhosphateH Symporter (PHS) Family
• 2.A.1.10 The NucleosideH Symporter (NHS)
  Family
• 2.A.1.11 The OxalateFormate Antiporter (OFA)
  Family
• 2.A.1.12 The SialateH Symporter (SHS) Family
• 2.A.1.13 The Monocarboxylate Porter (MCP) Family
  
• 2.A.1.14 The AnionCation Symporter (ACS) Family
  
• 2.A.1.15 The Aromatic AcidH Symporter (AAHS)
  Family
• 2.A.1.16 The Siderophore-Iron Transporter (SIT)
  Family

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Phylogenetic criteria
-Identify S.cerevisiae ORFs of TMS gt 2
-Identify TC using BLAST -Apply the
following general criteria -for 3th digit
(Super)Families 10 identity (E lt 10
20) -for 4th digit Subfamilies 20
identity (E lt 10 35) -for 5th digit Clusters
35 identity (E lt
10-65)
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• Explore Transport Protein Diseases.doc

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YETI YEAST TRANSPORTOSOME INFORMATION
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BLAST QUERY OF P39980 S.cerevisiae UMP
BLAST
FASTA
P39980 YEH5_YEAST (YEL065W)HYPOTHETICAL 70.6
KDA PROTEIN 1195 0.0 P38731 YHE0_YEAST
(YHL040C)HYPOTHETICAL 70.9 KDA PROTEIN
530 e-149 P38724 YHE7_YEAST
(YHL047C)HYPOTHETICAL 71.6 KDA PROTEIN 431 e-119
P36173 YK86_YEAST (YKR106W)HYPOTHETICAL 69.0
KDA PROTEIN 393 e-108 P25596 YCH0_YEAST
(YCL073C..)HYPOTHETICAL 68.9 KDA
PROTEIN 392 e-108 Q08299 ENB1_CHROMOSOME XV
READING FRAME ORF YOL158C 280 4e-74
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Subfamily 2.A.1.16
The Siderophore-Iron Transporters (SIT)
Clusters
Sit1/Arn3 (YEL065w) S.cerevisiae (spP39980)

The ferrioxamine H symporter
2.A.1.16.1
Enb1 (YOL158c) S.cerevisiae (pirS66857)

2.A.1.16.2
The ferric enterobactin H symporter
Taf1 (YHL047c) S.cerevisiae (spP38724)

2.A.1.16.3
The ferric triacetylfusarinine H symporter
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PLEIOTROPIC DRUG TRANSPORTERS (PDR) in YEASTS
also called Multiple Drug Resistance (MDR)
in MAMMALIAN CELLS
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The 16 "full - size" ABC transporters
The 16 "full - size" ABC TRANSPORTERS
PDR5 - like
MRP - like
Pdr12
Pdr12
Pdr10
Yor1
Ykr103/4
Pdr10
Yor1
Snq2
Snq2
Ykr103/4
Pdr15
Pdr15
Ynr070
Ynr070
Yhl035
Yhl035
Pdr5
Pdr5
Pdr11
Pdr11
Bat1
Bat1
Yor011
Yor011
Bpt1
Bpt1
Ycf1
Ycf1
Yol075
Yol075
MDR - like
MDR - like
NEW
NEW
(TM-NBF)2
(TM-NBF)2
(NBF-TM)2
(NBF-TM)2
Ste6
Ste6
Ste6
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The 16 "full - size" ABC
The 16 "full - size" ABC
PDR5 - like
PDR5 - like
MRP - like
MRP - like
Pdr12
Pdr12
Pdr10
Yor1
Ykr103/4
Pdr10
Yor1
Snq2
Snq2
Ykr103/4
Pdr15
Pdr15
Ynr070
Ynr070
Yhl035
Yhl035
Pdr5
Pdr5
Pdr11
Pdr11
Bat1
Bat1
Yor011
Yor011
Bpt1
Bpt1
Ycf1
Ycf1
Yol075
Yol075
MDR - like
MDR - like
NEW
NEW
(TM-NBF)2
(TM-NBF)2
(NBF-TM)2
(NBF-TM)2
Ste6
Ste6
Ste6
MULTIPLE disruptions
MULTIPLE disruptions
SINGLE disruptions
NO disruptions yet
SINGLE disruptions
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THE YEAST ABC TRANSPORTER PDR5 MEDIATES
RESISTANCE TO MANY DIFFERENT COMPOUNDS
Steroids and their structural analogues5??
androstan 3?? ol 17 one 5?? androstan 3?? ol 17
one 5?? androstan 17? ol 3 one 5 ??androstane
3,17 dione 5?? androstane 3,17 dione 1,4
androstadiene 3,17 dione 5 ??pregnan 3 ??ol 20
one 4 pregnen 20 ??ol 3 one trans
dehydroandrosterone 4 androstene 3,17
dione epiandrosterone acetate epiandrosterone estr
iol 16, 17 diacetate 17 ??ethynyl
estradiol deoxycorticosterone pregnenolone progest
erone testosterone diethylstilbestrol hexestrol 4
hydroxytamoxifen
Herbicides sulfomethuron methyl fluometuron chloro
xuron difenoxuron chlorbromuron diuron
Anticancer drugs chemosensitizers tamoxifen dauno
rubicin doxorubicin trifluoperazine
Fluorescent dyes rhodamine 6G rhodamine 123
Fungicides itraconazole ketoconazole econazole mic
onazole triadimenol bitertanol nuarimol fenarimol
carbendazim diclobutrazol phenapronil chlorothalon
il cyprodinil dodine imazalil prochloraz azoxystro
bin flusilazol tebuconazole CGA64251 2
mercapto- -benzothiazole krezoxim
methyl nystatin soraphen A epoxiconazole
Detergents tetradecyltrimethyl- -ammonium
bromide hexadecyltrimethyl- -ammoniumbromide zwitt
ergent 3-10 zwittergent 3-12 zwittergent
3-14 brij 58 brij 35 n octylglucoside n
dodecylglucoside n dodecylmaltoside
Ionophores valinomycin nigericin monens
in A23187
Antibiotics chloramphenicol tetracycline
anisomycinantimycin A1 cycloheximidelincomyci
noligomycin tunicamycinthiolutin
Others 2,3,5 triphenyltetrazoliumchloride 4
nitroquinoline n oxide 8 hydroxyquinoline diphenyl
amine p aminodiphenylamine n decylamine 4
nitrophenol aniline anthron tetranitrotetrazoliumc
hloride blue resazurine sporidesmin staurosporine
fluphenazine cerulenin compactin

PDR5
Kolaczkowski et al., 1997
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Pdrp are activated in antifungal-resistant
human and plant pathogens such as Candida
albicans ,Candida glabrata,Candida krusei,
Cryptococcus neoformans and others ??
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MIYAKAMA et al, 2005
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ECL3
ECL6
Balzi et al., JBC (1994)
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The catalytic Lys from NH2 Walker A is
substituted by Cys in Pdr sensu stricto
Linker region
Walker A
Walker B
GRPGSGCTT
Pdr5-NH2
GASGAGKTT
Pdr5-COOH
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Fig. 1
TMD1
TMD2
EL3
EL6
NBD1
NBD2
EL1
EL4
EL2
EL5
C
4
8
12
1
2
3
5
6
7
9
10
11
IL1
IL2
IL3
IL4
N
Walker A2 (GX2GXGK(S/T))
Walker B2 (hhhhD)
ABC 2 (LNVEQ)
Helical domain
Walker A1 (GX4GC(S/T))
Walker B1 (X3WD)
ABC 1 (VSGGE)
Helical domain
Q-loop (Q)
H-loop (H)
D-loop (E)
P-loop
C-loop
pro-loop (P)
Q-loop (E)
H-loop (Y)
D-loop (D)
P-loop
C-loop
pro-loop (X)
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Pdr Transportersonly in fungi, plants and
protists

• Sensu stricto Pdr (Pdr5/10/12/15/18, Snq2)
• Reverse topology
• Cysteine in N-terminal Walker A
• Expanded ECL3 and ECL6
• Efflux pump
• Honorary Pdr (Aus1, Pdr11, YOL075)
• Reverse topology
• Influx pump?

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Snq2
Sensu stricto Pdrp in SACE
YNR070w (Pdr18)
Pdr12
Pdr10
Pdr5
Pdr15
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COMPARATOMICS
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Hemiascomycetes
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2.A.5.Y2 no data no data
YALI-0D19008g
2.A.5.Y1 Golgi Mn
KLLA-0F17886g
DEHA-0E06105g
KLLA-0A07601g
2.A.5.2 ER Zn
SACE-YKE4
SACE-ATX2
CAGL-0K05577g
YALI-0F15411g
DEHA-0B16335g
YALI-0E00748g
YALI-0D00759g
DEHA-0E25388g
SACE-ZRT2
SACE-ZRT1
CAGL-0M04301g
YALI-0F21659g
KLLA-0D16434g
2.A.5.1 plasma membrane Zn
DEHA-0B07337g
CAGL-0E01353g
The Yeast ZIP Family (ZINC Iron Porters). TC
2.A.5.
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CONCLUSIONSREDONDANT TRANSPORTERS ARE LOCATED
IN DIFFERENT MEMBRANESSOME TRANSPORTER
SUBFAMILLIES HAVE NO SACE MEMBERS
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In practice the most variable families
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BLAST of Snq2 in SACE and KLLA
ScSnq2
ScYNR070
KlSnq2a
ScPdr12
KlSnq2b
ScPdr5
KlSnq2c
ScPdr15
ScPdr10
KlSnq2d
BLAST E-value is not discriminating BLAST score
distinguishes four Snq2 homologs in KLLA KlSnq2a
clusters with ScSnq2 KlSnq2b clusters with
ScPdr12 KlSnq2 c and d cluster with Sc Pdr5/10/15
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Neighbors analysisPdr5/15 filiation
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Basidiomycetae
Ustilaginom.
P
P
Tremellom.
P
Agaricom.
Homob.
Taphrinomycotina
P
P
Saccharomycotina
P
Ascomycetae.
P
P
P
P
P
Onygenales
P
P
Dothideomycetes
P
P
P
Pezizomycotina
P
P
P
P
P
P
Eurotiomycetes
(P)
P
P
P
P
Leotiomycetes
P
P
P
Sordariamyces.
P
P
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PHYLOGENETIC ANALYSIS OF 78 PDR (SENSU STRICTO
AND HONORAY)FROM 11SPECIES SACHARO/PEZIZO/BASIDIO
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PHYLOGENETIC CLUSTERS OF FUNGAL PDRs
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CONCLUSIONSA total of 8 clusters (A to
H)Three clusters contain only SACE members
(A,D,E)Two honorary clusters are conserved
(E,F)Four new clusters not present in
SACE(B,C,G,H) The four new clusters are sensu
stricto Pdrp (see Cys in Walker A1) Are the new
clusters drug efflux pumps? Which specificity?

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PHYLOGENETIC ANALYSIS of 349 Pdr from 56 species
distributed in 5 subphyla
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C
B
Fig. 3
E AUS1
H2
G - ----
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ConclusionOnly one more cluster H1/H2 that
belongs to sensu stricto PDR
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STRUCTURE PREDICTOMICS
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CONCLUSION 1.Combined to analysis of
mutants,the elucidated sequence motifs and the
prediced topology will become important for
interpretation of crystallographic data and for
elucidation of the catalytic mechanism of the
efflux pumps including the amazing pleiotropic
specificity for substrates and the asymetry of
the two NBD. 2.This infornation must be
taken into account to understand the molecular
evolution of this important protein family (gt500
millions years?) 3.This molecular
information should facilitate the design of more
sensitive antifungals.
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• COLLABORATORS
• OTAGO
• MONK brian
• CANNON richard
• NIMMI kyoko
• LAMPING erwin
• LLN/GENA
• BARET philippe
• DE HERTOG benoit
• DIFFELS julie
• SERET marie line

• LLN/FYSA
• BALZI elizabetta
• KOLASCZKOWSKI marcin
• SAN DIEGO
• SAIER milton

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ACKNOWLEDGEMENTS
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BIBLIOGRAPHY REVUES PDRMonk B.and Goffeau A.
2008, SCIENCE 321367-369 Richard Cannon et
al. 2009,CLINICAL MICROBIOLOGY REVIEWS 22291-321
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Phylogeny of human fungal pathogens
Coccidioides immitis
Coccidoides-Histoplasma Cluster
Histoplasma capsulatum
Aspergillus nidulans Aspergillus fumigatus
Aspergillus Cluster
Aspergillus oryzae
Aspergillus terreus
Candida lusitaniae Candida guillermondii
GTG
Candida parapsilosis Candida tropicalis Candida
dubliniensis Candida albicans
Candida Cluster
Candida glabrata
Saccharomyces Cluster
Saccharomyces cerevisiae
WGD
Pneumocystis carenii
Pneumocystis Cluster
Cryptococcus neoformans
Cryptococcus Cluster
CHYTRIDS
Chytridium comfervae
ZYGOMYCETES
Rhizopus oryzae
Homo sapiens

1000 800 600 400
200 0
Millions of years
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Snq2p in Hemiascomycetes
RO1395
RO1393
RO1392
RO1387
RO1386
RO1385
RO1382
Z. rouxii
TH11581
TH11579
TH11568
TH11564
TH11563
TH11559
TH11558
K. thermotolerans
WA12069
WA12062
K. waltii
WA12924
WA12921
WA12920
WA12917
WA12916
K. lactis
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