Characteristics of CRE1 cytokinin receptor

1000 aa long N terminal ligand binding domain ( 300 aa) Histidine kinase domain at the C terminus

followed by two receiver domains T 278 mutation causes the loss of function A part of the two component system

Thr 278

HK R RLB

TM

Histidine kinases

CYCLASES/HISTIDINE KINASES ASSOCIATED SENSORY

EXTRACELLULAR /CHASE/

200 – 230 aa Present in bacteria, lower eukaryotes and plants Bound at the C terminus Binds cytokinins and peptides Helices at the both ends Two internal helices divided by strands (a + b

fold) Mechanism of ligand binding and evolutional

relationships are unknown.

Evolutionary relationships to CHASE domain

(GRDB-Gene Related Data Base)

1jogA - sensory domain of the membraneous two-component fumarate - sensor Dcus from E. coli, 1p0zA - sensor kinase cita. 1e4eA, 1ehiA - D-Alanine-D-lactate ligase.

Structures of receptor domains selected by 3DHit as related to

1joga.

Domain 3d-hit score

PDB code

Function Organism Ligand

CACHE 84.3 1p0z Ca2+ channels and chemotaxis receptors

K.pneumoniae Citrate Anion

GAF 55.6 1mc0 cGMP phosphodiesterase, adenyl cyclase, FhlA domain

M.musculus Cyclic guanosine monophosphate

PAS/PYP 47.2 1f98 Periodic clock protein, aryl hydrocarbon receptor and single-minded protein/Photoactive Yellow Protein

E. halophila 4'-Hydroxycinnamic Acid

Profiln 46.2 1g5u monomeric actin binding H. brasiliensis Actin ACT - 1psdA1 Aspartate kinase –

chorismate mutase – TyrA E.coli Nicotinamide-

Adenine-Dinucleotide

Topological representation and evolution of PYP family

Model of A. thaliana CRE1a receptor with trans-zeatin and kinetin

Conclusions

CRE1 and 1mc0 are located on the same clade and may have common origin

Cre1 is lacking a strand they are structurally much more similar to each other then to other members of PAS/PYP superfamily

The ligands bound by both domains are also structurally similar

CACHE, PAS/PYP and Profilin form a separate branch. These two groups separated very early in evolution.

Most of the protein-ligand interaction fulfiled by closely related receptors which are diverse in primary structure but share a common structure.

S-RLK LRR-RLK C4-class(TNFR)

WAK(EGF)

PR5-RLK Lectin-class

S-domain LRR-domain Kinase-domain Lectin-domain

TM-domain

RCC1-like repeat

TNFR-like repeat

EGF-like repeat

PR5-like domain

Human skin fibroblasts can be grown in zeatin (40 to 200 µM) throughout their lifespan of about 300 days in the lab. It neither decreases nor increases the division potential of human cells.

Long term growth

Implications• Zeatin has no short-term or long term negative effects on human cells.

•Zeatin does not interfere with the genetic control of cellular lifespan, hence no danger of cancer induction.

Summary

1. Short-term or long-term treatment of human skin fibroblasts with zeatin (optimal dose 80 µM) has no toxic effects in terms of cell survival, growth, and lifespan.

2. Zeatin does not induce extra cell proliferation in human cells, and thus, it is not carcinogenic.

3. Zeatin treated cells maintain youthful characteristics in terms of morphology, cell size and actin cytoskeletal organization.

4. Zeatin treatment enhances the antioxidative enzyme activities of human cells against oxidative damage.

5. Zeatin treatment increases the stress tolerance ability of cells.

A time scale of radiolytic events in biological system

Interaction Time (sec) Events and Processes

Physical 10-18 - 10-15 Energy absorption, excitation and ionization

Physico-chemical 10-15 - 10-10 Rearrangement of ionized or excited molecules, formation of diffusible radicals such as •H, •OH, e-

aq

Chemical 10-10 - 10-3 Free radical reactions, molecular alterations, formation of bioradicals by indirect action. long lived lesions in macromolecules

Biochemical 10-3 - 104 Enzymatic reactions, recognition of lesions, repair, fixation of damage

Cellular level 104 - 107 Cell death, cell loss, division kinetics, mutation

Systemic(multicellular organisms)

108 - 1010 Hormonal effects, immune reactions, vascular changes, functional impairment, adaptation, carcinogenesis, ageing, death

Some of the events and processes are overlapping. The times indicated are approximate.

N - B enzy lade nine6

N - B e n z y l a d e n i n e6

00 2 0

2 84 3

5 3

8 1 1 8 6

2 1 5

5 7 6 99 6

1 0 81 1 9

1 4 9

1 6 0

1 7 1 1 9 81 3 5

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0

5 0

1 0 0

2 0 00 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

w a v e l e n g t h [ n m ]

AB

SO

RB

AN

CE

2 2 0 2 4 0 2 6 0 2 8 0 3 0 0

N N

O

NH

N

C H 2

N H

A B

C

D

K i n e t i n

Model of CHASE domain with docked zeatin

Model of CHASE domain with docked kinetin

Adenine binding site in the crystal structure of Viscum album lectin I

Adenine binding pocket in the crystal structure of Viscum album lectin I

Kinetin binding site in crystal structure of Viscum album lectin I

Kinetin binding site in crystal structure of Viscum album lectin I

Crystal structure of the catalytic pocket of mistletoe lectin I (Viscum album) with kinetin

Zeatin binding site in the crystal structure of Viscum album lectin I

Crystal structure of the catalytic pocket of mistletoe lectin I (Viscum album) with trans-zeatin

KINETIN(N6-Furfuryladenine)

� Naturally occurring plant growth factor (cytokinin)

Anti-Aging Mechanisms:� Retards senescence in plants� Anti-aging effects in human skin cells in culture

-slow & reverse alternations that naturally occur in cell-aging process� Powerful antioxidant (free radical scavenger-protection)

KINETIN(N6-Furfuryladenine)

� Plant growth factor (cytokinin)� Retards senescence in plants� Anti-aging effects in human skin cells in culture� Antioxidant (free radical scavenger)(Protection)

The Future of Cytokinins in Skin Care and Anti-Aging

New clinical applications of Kinetin ZEATIN – New product development In vitro screening for new cytokinins Mechanisms of action

MODIFIEDBASES

MALONDIALDEHYDE

(Tyr, Phe, Trp, Met, Cys, Ser, Lys)

NUCLEICACIDBASES

LIPIDS

PROTEINS

SUGARFURFURAL

PROPENAL

OH.

DNA

00 2 0

2 84 3

5 3

8 1 1 8 6

2 1 5

5 7 6 99 6

1 0 81 1 9

1 4 9

1 6 0

1 7 1 1 9 81 3 5

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0

5 0

1 0 0

2 0 00 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

w a v e l e n g t h [ n m ]

AB

SO

RB

AN

CE

2 2 0 2 4 0 2 6 0 2 8 0 3 0 0

N N

O

NH

N

C H 2

N H

A B

C

D

K i n e t i n

0.853

H2O1.527

0.716

O31.549

O2

H3O

0.993H2

1.516

1.520P1

0.903

H10A1.320

1.340

C21.357

N1

0.943

H10B

1.054

O4

1.358N3

1.469

1.483

C10

O1

H4O

1.411

1.337C6

0.953N61.374

1.369

C9

1.378C5

1.298 1.322C11

H6

H131.366

C13

1.330

0.808

N9H9

1.480

O15

1.332

0.935N7

0.934

C8

H7

1.252

1.058C12

H12

0.954

C14

H81

H14

a

bc

POCNH

N - B enzy lade nine6

N - B e n z y l a d e n i n e6

b

c

POCNH

 A time scale of radiolytic events in biological system

 

Interaction Time (sec) Events and Processes

Physical 10-18 - 10-15 Energy absorption, excitation and ionization

Physico-chemical 10-15 - 10-10 Rearrangement of ionized or excited molecules, formation of diffusible radicals such as •H, •OH, e-aq

Chemical 10-10 - 10-3 Free radical reactions, molecular alterations, formation of bioradicals by indirect action. long lived lesions in macromolecules

Biochemical 10-3 - 104 Enzymatic reactions, recognition of lesions, repair, fixation of damage

Cellular level 104 - 107 Cell death, cell loss, division kinetics, mutation

Systemic(multicellular organisms)

108 - 1010 Hormonal effects, immune reactions, vascular changes, functional impairment, adaptation, carcinogenesis, ageing, death

   Some of the events and processes are overlapping. The times indicated are approximate.

2 3 4 5 61

K

A

( R f = 0 . 7 6 )

2 3 4 5 61

K

A

( R f = 0 . 7 6 )

500 g DNA (herring sperm) → 275 mg KC.O. Miller et al. J. Amer. Chem. Soc. 78: 1375-1380 (1956)

22 mg DNA (salmon testes) → 12 µg I. Gawronska, 2005 – unpublished

Yield 0.2 %