Comparison of the BTB Superfamilies in Physcomitrella ...

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Comparison of the BTB Superfamilies in Physcomitrella patens and Arabidopsis thaliana

Jacob P. Janssen, Dr. Derek Gingerich,University of Wisconsin-Eau Claire

K

Target

BTBMotif

CUL3

RbxUb

C E2

BTB

The Arabidopsis and Physcomitrella BTB families encoded many similar BTB proteins

Figure 2. Grouping of the BTB subfamilies based on the nature of additional motifs outside of the BTB domain along with a protein diagram of representative members.

135987

2261782345479872811485710661652205

At5g64330

NPR1At3g48360

At5g19330At5g55000

NPH3

Ankyrin

TPRArmadilloPentapeptideOther

30At3g43700MATH 6

75321

26

17363

PhyscomitrellaArabidopsis

225

Other27

1 0 6 6 1 6 1 S DV T F L - V E GR R F Y AHR I AL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L AS S DAF R AMF D- - GG2 2 6 1 7 8 1 ADV K F E - V DGE L Y HAHK L V L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AAR S P V F K AQL F - - GP1 7 0 4 5 1 1 S DV V F E S L DGR K V Y GHR AV L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AS QS DV F K AMF S - - MP2 3 1 8 9 4 1 V DV E L QGV DGDR V HAHK AI L S V GWV L V DP S L C S E L GQV L L S T I HE S V R R R C P Y V I AE K K S V AS K S I E F E AMF R - - AD1 3 0 7 6 9 1 T DV T I C S AT G- C V GAHR AI L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AAR S P V F DS MF S - - HN1 6 5 6 6 0 1 ADV C F L - V GNE K F R C HR F V L G- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AR S E Y F K AR F S R T T G1 8 9 9 2 2 1 MDT S V V - L R S K T I HI S S AI L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AAK S P Y F Y K L F S - - NG9 8 7 2 8 1 ADL T V C T DDGS QI HV HS MV L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MAS S P V F K F Y L Q- - K E1 1 4 8 5 7 1 NDI V F H- L GGDAV P C NR E K I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AGL S MP F NT ML N- - GV1 3 5 9 8 7 1 S DI V V E - AGGMNF S L HK F P L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V AR S GR I R K L V ANL AD1 8 7 7 0 7 1 - S V V HL NV GGMMF AT T V DT L T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QR DT DS ML AV MF S GR HR1 0 4 5 4 5 1 - GR AK I E V GGR V F V T T MQT V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E K AGK DS HL Y K - - - T

1 0 6 6 1 6 K R L C E Y S MAQNL -2 2 6 1 7 8 R L L C E S K L C E NV -1 7 0 4 5 1 K S L C E QE I L QS L -2 3 1 8 9 4 L E V V C E DS L V K NM1 3 0 7 6 9 K E AC E S S L V DDI -1 6 5 6 6 0 K R AV T DAL L P QL -1 8 9 9 2 2 MR Y C S - R L L K NMP9 8 7 2 8 K QT C T V HF E QGL -1 1 4 8 5 7 K DAC DQNL AT F V -1 3 5 9 8 7 V AR T E AF L S E V V -1 8 7 7 0 7 V E NI T P F L C K K D-1 0 4 5 4 5 L R NAHGR AP L NG-

1 0 6 6 1 62 2 6 1 7 81 7 0 4 5 12 3 1 8 9 41 3 0 7 6 91 6 5 6 6 01 8 9 9 2 29 8 7 2 81 1 4 8 5 71 3 5 9 8 71 8 7 7 0 71 0 4 5 4 5

Y R E K E A- - - - - - - - - - - L DI E I P MR DR NS - - - - - - - - - - - DHI E I K MAE GT P - - - - - - - - - - - L HME MN T DR K V V - - - - - - - - - - - WNL E MP L ME K QS - - - - - - - - - - - AT V R I N F R E GV AGL AT NS GADS L L I L QE N MR E S E Q- - - - - - - - - R DV T L R I T V R K S R F P - - - - - - - - - - - AI DI F F L E AR MC D- - - - - - - - - I GF S K N S E NP NL - - - - - - - - - - - L QL T DV L HMDS K - - - - - - - - - - - E GAV F I V S QS GQ- - - - - - - - - - - - - - GWF

NI S WK V F E L MMR F I Y - - - - - - - - - - T GNV DMAT DNAQDL L R AADQY L L EDI E P P V F K AL L HF I Y R DS L P DT K E L I GAP S T S T L L AQHL L AAADR Y GL D- - - Y P AL DAF I F F F Y R - - - - - - - - AT V NQAV L R NHL V E L L QAADK Y GI E- - - Y V AL K AF V T F F Y T - - - - - - - - GR V S S S V L I E Y L T T L L DAAHK Y NV QDMC L E S C R AL L S F L Y G- - - - - - - - - NL K Y P DF R K HR V AL V R AAHK Y DI VDL S AS AF E K V L E Y I Y T - - - - - - - - - DS V K T V DL DE AE E L F DAAS R Y L L FQAE E S P L MDL L QF MY S - - - - - - - - - GR V QANT P AT V L DV L MAADK Y E V AGV P F HAV R C F L R Y L Y S - - - - - - - - S R C E R S DME E C AL HL V V L AHT Y MV PGI S V T GMR AV DHF S K T - - - - - - - - - GR L AR L S P E ML L E I L S F ANR F C C DP GGAE AF DL AAK F C Y G- - - - I NF E I T T S NV AV L R C AAE Y L DMT E S Y GE NDR DGT HF R HI L NWL R D- - - - - - - - - GV I P ML E I S AY QE L HR E AE Y Y QL MDR DP DMF NI L L NML R T G- - - - - - - V V Y P K P E S L GF L DR L I E E AT F Y GI E

GRAFDPTATNGE

LLL

LLCLLLLC

-

Sequence Alignment of Representatative Physcomitrella BTB Domains.

Introduction Following their synthesis, proteins in cells can be modified in a variety of ways. These modifications may modulate the protein’s activity, localization, interactions with other molecules, or stability. One such modification is the covalent attachment of a small globular protein called ubiquitin (Ub). Ubiquitination can change the activity or localization of a protein, if one Ub moiety is attached, or can lead to degradation of the protein if chains of Ubs are added. Selective degradation of key proteins by ubiquitination has been shown to be an important regulatory mechanism in many signaling pathways in eukaryotes. The attachment of Ub to target proteins is catalyzed by E3 Ub-ligases. One family of ligases are the BTB/Cullin 3 (CUL3) E3s. These complexes are composed of three proteins: CUL3, which acts as a backbone, RBX, which helps recruit the Ub, and a BTB (Bric-a-Brac/Tramtrack/Broad Complex) domain-containing protein (Fig. 1). The BTB motif allows the BTB protein to bind CUL3. Besides the BTB motif, BTB proteins contain other sequences (such as MATH or NPH3 domains) which recruit the target. BTB gene families are very diverse in eukaryotes. The sets of BTB proteins encoded in animal genomes are very different from those encoded in plants, for instance. We are interested in how the BTB gene family has changed during plant evolution. Dr. Gingerich has previously characterized the BTB family in a dicotyledenous flowering plant, Arabidopsis thaliana. In this studied, I have begun to characterize the family in a simple nonvascular moss, Physcomitrella patens. Significant differences in the BTB families might suggest that the two plants use the BTB/CUL3 E3s to target different proteins for degradation.

Conclusions

9 9

1 3 71 0 0

7999

1 0 2 1 0 2 1 0 2 1 0 2 1 0 8 1 0 4

79

BA1

D2RL

BPECJ

A2A2

39 known eukaryotic BTB domains used as initial queries

69 loci identified with < 0.0004 E-value

analysis and reannotation of gene sequences4 loci predicted to be nonfunctional

65 loci identified with < 0.0004 E-value

Figure 1 BTB/Cullin 3 E3 Ubiquitin-Ligases

77289

93266 61156

118125

130769

66017

97923

74560 124356150882

85405

87316

50789

42628 159693

219111

134565

30287

164153

100

100

100

100

93

100

6365

100

97

35

94

78

100

100

100

100

100

82

100

100

69100

10077

95

32

39

53

90

52

99

100

100

58

45

100

92

56

100

96

96

99

90

67

35

72 56

33

29 43

205 6

9

24 6

1

6

0

11

NPH3TAZ-CaM B DTPR

Armadillo

PentapeptideAP2Back 3

Physcomitrella ArabidopsisSubfamily size

626

3053

1

1380

Scop Domain 162

51

0

"A2" 43"D2" 21"F" 30"H" 56

Ankyrin 170451

106616

106476

165660

143618

226178

217835 98728

150321

223881

234547

231894 189922

122867

3260

121620 119190

31431

67811

231552

93641

41851

154574

21814

135987 127110

134520

12061 106949

12163

31592

2869 1322

22014

161287

114857

80508

50218

167201

111151

104545

148281

125607

187707 52205

52179

173

3

2

MATH

Other 5

00

Total 65

1

RPT2

NPH3, RPT3

PRL1-IFG

NPR1

AtPOB1

AtBT5AtBT4

AtBT3AtBT2AtBT1

At5g21010At3g43700

At3g03740At2g39760At5g19000

At3g06190At5g55000

At5g41330At3g09030At4g30940At2g24240

At5g19330At5g13060

At4g02680 EOL1At5g58550EOL2

At3g51770 ETO1At2g05330

At5g48510At4g04090

At2g40450At3g29740

At2g40440At3g56230

At1g01640At1g55760At1g21780

At4g08455

At4g37590 At2g23050At5g67440 At2g14820

At4g31820At5g47800

At1g67900At3g26490

At3g44820At5g10250

At3g50840At5g66560

At5g64330At1g30440

At5g03250 At5g13600At3g49970

At5g67385At5g48130

At2g30520At3g08570

At3g08660

At5g63160At3g48360

At1g05690At5g67480At4g37610

At2g30600At4g01160

At3g61600At2g46260

At5g45110At4g19660

At4g26120At1g64280

At3g57130At2g41370

At5g60050At1g63850At3g50780

At2g13690At3g05675

At5g48800At1g03010

At2g47860At3g22104

At1g50280At3g19850

At3g49900At3g03510

At5g17580At2g04740

At1g04390

10099

85

81

65

48

10026

4626

11100

9971

5213

933229

91

97

99

4646

37

42

6

100

9954

98

97

10096

69100

100

090

9760

58

2

5100

99

40

91100

6643

2799

2810

3

101997

3326

12

811

2851

100

79

7561

8586

8078

100

100

ARIA

BOP1BOP2

Phylogenetic Analysis of the BTB Superfamilies in Physcomitrella and Arabidopsis

Physcomitrella

Arabidopsis

Identification of BTB domain-containingproteins encoded by the Physcomitrella genome

BLAST search Physcomitrella patens genome v1.1 predicted protein sequences (from the DOE Joint Genome Institute Physcomitrella genome sequencing project)

Domain # of Genes Protein ID

TAZ zinc fingerETO1

# of Genes Protein ID

BA1

D2RL

BPECJ

A2A2

BA1

D2RL

BPECJ

A2A2

Figure 3. Sequence alignment of representative Physcomitrella BTB domains. The approximately 100 amino acid core BTB sequence from 12 representative Physcomitrella BTB proteins were aligned with ClustalW and displayed with Boxshade using a threshhold of 55%. Identical and conserved amino acids are shown with black and grey, respectively. Dashes denote gaps. Desginations to the left indicate the BTB family and the JGI number for each protein.

Some data presented in this poster was generated by Dr. Gingerich when he was funded by an NIH NRSA post-doctoral fellowship (#GM068361)

100

43

00

“K”“Q”

JCSTA2

Q I G H P R MN E A1

B L D2

K

J I H G F E D2

D1

C B A2

A1

Figure 4. Phylogenetic trees of the complete BTB protein superfamilies from Arabidopsis and Physcomitrella. The approximately 100 amino acid core BTB domain from all Arabidopsis and Physcomitrella BTB proteins were aligned byClustalW. The alignments were used to generate non-rooted phylogenetic trees with MEGA 4.0, using the Poisson distance method and a bootstrap value of 1000. The subfamilies identified from the phylogenetic analysis are marked on the bottom of the trees. Individual members of the tree are color-coded by the nature of the domain(s) either N- or C-terminal to the BTB domain. Where possible, designations for proteins previously identified by other methods are used.

-Physcomitrella patens encodes 65 putative BTB domain-containing proteins in its genome.

-There are a variety of different motifs/domains attached to BTB domains in the PhyscomitrellaBTB family, suggesting a number of different types of target proteins.

-The Physcomitrella and Arabidopsis BTB families are similar in overall size and share many similarBTB types, suggesting the two plants use the BTB family in similar ways.

-However, there are also several Physcomitrella and Arabidopsis-specific types, suggesting some specialization in each lineage.

-The BTB-NPH3 family in Arabidopsis is significantly larger than that in Physcomitrella (30 vs. 17), suggesting that there may have been an expansion of this subfamily as more complex plants evolved.