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Stazione Zoologica Anton Dohrn. MODO ( MOD el O rganisms) fellowship. Identification of thyroid hormon precursor(s) in Ciona intestinalis endostyles. Maria Salzano. Tutor: Doct. Antonietta Spagnuolo Cellular and Developmental Biology Group. Satoh, 2003.

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slide1

Stazione Zoologica Anton Dohrn

MODO (MODel Organisms) fellowship

Identification of thyroid hormon precursor(s)in Ciona intestinalis endostyles

Maria Salzano

Tutor: Doct. Antonietta Spagnuolo

Cellular and Developmental Biology Group

Satoh, 2003

MODO meeting: 27th of June 2013

slide2

The link between Endostyle and Thyroid

Anatomical and molecular studies confirmed that a THYROID-LIKE FUNCTION lies within the ENDOSTYLES also of Urochordates and Cephalochordates. (Ogasawara et al., 1999)

Specific endostyle cells have been shown to concentrate radioiodine from sea water (Barrington, 1957), and to produce monoiodiotyrosine (MIT), diiodiotyrosine (DIT) and thyroxine

(Roche et al., 1959); (Kennedy, 1966); (Dunn, 1974).

Thyroidhormones play a criticalrole in amphibians[Furlowet al.,2006; Tata, 2005], urochordate[Patricoloet al., 2001], amphioxus[Paris and Laudet, 2008] and lamprey[Manzonet al., 2001] metamorphosis.

In vertebrates

TG is the protein precursor of T3 and T4

PROJECT AIM

Identification of the protein(s) used

as scaffold for THs biosynthesis

in Ciona genome, no homologus of TG gene has been found

slide3

V

D

As negative control..

Preliminary results

Free T4

Endostyles total

proteins extracts

LC analysis on column

Proteins are dried and digested (pronase)

T3/T4 bound to protein/s analysis

Proteins are removed (ice EtOH; -20°C)

free T3/T4 analysis

* in collaboration with

A. Amoresano’s group

Selected Reaction Monitoring

mass spectroscopy analysis

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Preliminary results (2)

PEPTIDE IDENTIFICATION

Software for identification, characterisation and quantization of proteins using mass spectrometry data.

Proteins are dried and digested (pronase)

T3/T4 bound to

Protein analysis

* in collaboration with A. Amoresano’s group

slide5

A parallel strategy…

IP

anti T4-Ab

Sup

NB

Endostyles TE

Endostyles total proteins extracts immunoprecipitated with anti T4-Ab

IP

Anti T4-Ab:

Sup

NB :

Supernatant of proteins Not

Ab - Bound

Protein Immuno – Precipitated with anti T4-Ab

Endostylus : TE

Endostyles proteins Total Extract

75 Kda

50 Kda

37 Kda

25 Kda

*

* in collaboration with Prof. Pucci’s group

slide6

Next analysis

Selected Reaction Monitoring mass spectroscopy analysis

1)

2)

blood

+ KClO4

Is there T4?

Is T4 free or bound to proteins?

What are the effects on T4 production in endostyles??