TRISTETRAPROLIN:ITS ROLE IN INFLAMMATION AND

CANCER

HIMADRI NATH

SEM -III, M.Sc. ZOOLOGY

ROLL:BGC/ZOOL NO:14101

BARASAT GOVT. COLLEGE

TRISTETRAPROLIN

1. Tristetraprolin (TTP), also known as zinc finger protein 36 homolog

(ZFP36), is a protein that in humans, mice and rats is encoded by the

ZFP36 gene. It is a member of the TIS11 (TPA-induced sequence) family.

2. In humans, ZFP36 gene is located on chromosome 19q13.1 , and consists

of two exons and one intron.

3. TTP protein levels are normally low and predominantly nuclear in

quiescent cells, whereas mitogenic stimulation of cells induces TTP

expression and promotes rapid TTP translocation from the nucleus into the

cytoplasm.

4. The two zinc fingers of TTP are required for its nuclear import, export of

TTP from the nucleus requires a nuclear export sequence (NES) present at

the amino terminal end of the protein.

FUNCTION OF TRISTETRAPROLIN

1. Tristetraprolin (TTP) is one of main mRNA destabilizing proteins. TTP

is an AU-rich element-binding protein (ARE-BP) belonging to a zinc-

finger family of proteins with a Cys-Cys-Cys-His (CCCH).

2. Adenosine and uridine (AU)-rich elements (ARE),sequence

UUAUUUAUU, in the 3′UTR of the m RNA serve as binding site for

Tristetraprolin.

3. The site of RNA binding is the central tandem zinc finger domain (TZF)

of TTP.

4. The ARE-binding activity of TTP was shown to be dependent on the

CCCH residues of TTP’s two zinc-finger domains and a single cysteine

to arginine mutation in either of the zinc-finger domains attenuated ARE

binding.

5. Such binding of TTP with m RNA leads to cytoplasmic degradation of

m RNA as follows.

CYTOPLASMIC m RNA DEGRADATION

1. Deadenylation is thought to be the initial step in mRNA decay in all eukaryotes.

2. Vertebrates express at least three deadenylation complexes: the Ccr4/Caf1/Not

complex, the Pan2/Pan3 complex, and the poly A-specific ribonuclease (PARN)

complex .

3. Following deadenylation, transcripts can be rapidly degraded via the 5′-3′ Xrn1

exonuclease pathway or the 3′-5′ exosome pathway.

CYTOPLASMIC m RNA DEGRADATION

4. In the 5′-to-3′ decay pathway, the mRNP rearrangement that is brought about

by poly(A) tail shortening facilitates the recruitment of the decapping enzymes

Dcp1 and Dcp2 to the cap structure, resulting in message decapping .

5. Once decapped, the transcript is degraded by the Xrn1 5′-to-3′ exonuclease.

6. 5′-to-3′ decay components, including Dcp1, Dcp2, and Xrn1, are concentrated

in cytoplasmic granules called processing bodies (P-bodies).

7. In the 3′-to-5′ decay pathway, transcripts are fully deadenylated and then

degraded by the exosome 3′-to-5′ exonuclease complex.

8. 5′-3′ decay pathway predominates for most mRNA decay and most ARE-

dependent mRNA decay.

CYTOPLASMIC m RNA DEGRADATION

ROLE OF TRISTETRAPROLIN IN INFLAMMATION

1. For a successful yet controlled immune response, cells need to

specifically destabilize inflammatory mRNAs but prevent premature

removal of those still used.

2. The expression of genes TNF-α , GM-CSF , Cyclooxygenase -2, some

Interleukins (IL-2,IL-3,IL-8,IL-10) occur during inflammation.

3. The m RNAs of TNF-α , GM-CSF , Cyclooxygenase -2, some

Interleukins (IL-2,IL-3,IL-8,IL-10) contains (AU)-rich elements

(ARE), often located in the 3′ untranslated regions (3′UTR).

4. The sequence UUAUUUAUU, (ARE) in the 3′UTR of these m RNA

serve as binding site for Tristetraprolin.

ROLE OF TRISTETRAPROLIN IN INFLAMMATION

5. TTP now can rapidly degrade the m RNAs by recruiting exonuclease

or exosome ,which if followed by termination of Inflammation.

6. TGF –β is a pleiotropic cytokine plays a critical role in modulating

immune response and inflammation. It is reported that TTP expression is

upregulated by TGF-β, suggesting a potential role of TTP in mediating

the immunosuppressive action of TGF-β in vivo.

7. So, for proper termination of inflammation mediated by TTP binding

and subsequent degradation of m RNA coding for inflammatory

components.

REGULATION OF TTP EXPRESSION DURING

INFLAMMATION

1. A mechanism must be in place that actuates the inflammatory mRNA

degradation with some delay after the initial inflammatory stimulus.

The delay is variable but specific for individual mRNAs.

2. TTP- and p38 MAPK-driven negative feedback system that executes

delayed yet robust elimination of a large number of inflammation-

induced mRNAs in a qualitative and temporally resolved manner.

3. TTP can be phosphorylated by MAPK-activated protein kinase 2

(MK2), a downstream target of p38,at serines 52 and 178 in mouse,

60 and 186 in humans.

4. The serine-threonine phosphatase PP2A has been established as

promoting TTP dephosphorylation.

REGULATION OF TTP EXPRESSION BY

PHOSPHORYLATION AND DEPHOSPHORYLATION

REGULATION OF TTP EXPRESSION DURING

INFLAMMATION

5. TTP target binding likely occurs in the cytoplasm following the

pioneer round of translation.

6. When p38 activity is low and TTP is relatively unphosphorylated at

serines 52 and 178, TTP can bind to Not1, recruiting the Ccr4/Caf1/Not

deadenylation complex. This results in shortening of the poly (A) tail

and in decay of the TTP bound mRNA. The TTP protein itself is

unstable in the dephosphorylated form and is turned over by the

proteasome.

7. Upon signal-dependent activation of p38 and ERK, TTP proteins are

phosphorylated, inhibiting the interaction of TTP with Not1, and thus

recruitment of the Ccr4/Caf1/Not deadenylation complex.

REGULATION OF TTP EXPRESSION DURING

INFLAMMATION

8. Then TTP protein become resistant to proteasome degradation as it

get associated with 14.3.3 protein.

9. As the activating signal wanes, the phosphatase PP2A promotes

dephosphorylation of TTP, allowing resumption in decay activity.

10. Finally, a recent paper suggests that the ARE binding protein HuR

needs to replace TTP in order to TNF translation to occur .

In vitro binding assays demonstrated that HuR was able to displace TTP

from an ARE-containing RNA probe when TTP was phosphorylated by

MK2.

REGULATION OF TTP EXPRESSION DURING

INFLAMMATION

ROLE OF TTP IN CANCER

1. Several lines of evidence have indicate that loss of TTP expression

is a consistent feature in a variety of human malignancies, and

reexpression of TTP induces cell type-specific growth inhibitory

effects, implicating TTP as a potential tumor suppressor.

2. Vascular endothelial growth factor (VEGF) ,an angiogenic cytokine.

Overexpression of (VEGF), have been reported from tumors

originating from glial cells, breast cancer ,cervical cancer, colon

cancer.

3. It is reported that VEGF m RNA contains ARE region at its 3’ UTR ,

so this region can serve as a binding site for TTP .

4. When TTP expression induced in cancer cell lines , there is a

significant drop in VEGF expression also observed .

TTP as a therapeutic target of cancer

GLIOMA

1.Glioma shows over expression of vascular endothelial growth factor (VEGF)

and interleukin-8 (IL-8)

2.VEGF and IL-8 m RNA s stabilized by HuR.

3.Induced expression of TTP have growth inhibitory effects through

destabilization of VEGF and IL-8 mRNAs.

BREAST CANCER1. It has reported that micro RNA (miR 29a) has a role to develop drug

resistance.

2. Over expression of this microRNA in breast epithelial cells suppressed TTP

expression.

3. Restoring TTP expression to physiological levels subsequently led to reduced

cell proliferation and invasion of metastatic breast cancer cells.

TTP as a therapeutic target of cancer

CERVICAL CANCER1. Cervical cancer caused by Human Pappiloma Virus (HPV) .

2. Viral oncoproteins E6 and E7 to inactivate the p53 and pRB tumor

suppressor pathways, respectively .

3. TTP targeting of E6-AP mRNA for decay resulted in p53 protein

stabilization leading to cellular senescence.

COLON CANCER1. TTP expression in various stages of colorectal cancer has shown that loss-of-

TTP and gain-of-HuR functions in colon cancer are required to

promote expression of COX-2.2. TTP in colon cancer cells attenuates cell proliferation, and inhibits COX-2

expression.

3.So TTP can antagonize HuR-mediated over expression of COX-2 .

REFERENCES

1. Brooks S.A, Blackshear P.J., Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim Biophys Acta. 2014;1829(0): 666–679.

2. Brooks S.A , Connolly J.E , Diegel R.J ,Fava R.A , and Rigby F.C, Analysis of the Function, Expression, and Subcellular Distribution of Human Tristetraprolin . Arthritis & rheumatism.2002; 46: 1362–1370.

3. Sanduja S, Blanco F.F, Young L.E, Kaza V, Dixon D.A, The role of tristetraprolin in cancer and inflammation. Front Biosci.2012; 17: 174–188.

4. Kratochvill F, Machacek C, Vogl C, Ebner F, Sedlyarov V, Gruber AR, Hartweger H, Vielnascher R, Karaghiosoff M, Rulicke T, Muller M, Hofacker I, Lang R, Kovarik P. Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation. Mol Syst Biol. 2011; 7:560.

5. Schott J, Stoecklin G. Networks controlling mRNA decay in the immune system. Wiley Interdiscip Rev RNA. 2010; 1:432–456.

REFERENCES

6 Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol. 1999; 19:4311–4323.

7. Cao H, Deterding LJ, Blackshear PJ. Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin. Expert Rev Proteomics. 2007; 4:711–726.

8. Clement SL, Scheckel C, Stoecklin G, Lykke-Andersen J. Phosphorylation of tristetraprolin by MK2 Impairs AU-rich element mRNA decay by preventing deadenylase recruitment. Mol Cell Biol. 2011; 31:256–266.

Sincere thanks to Dr. Debjani Sarkar for providing guidance and important

suggestions for this presentation .

Thanks to Dr. Debojyoti Chakrabarty, the Head of the P.G. Dept. of

Zoology, Barasat Govt. College for providing infrastructure facilities.

Thanks to Dr. Tuhin Kumar Saha, Dr. Madhumita Manna,

Dr. Sanjay Podder, Dr. Srikanta Guria, Dr.Tanaya Dey for providing

encouragements and various help during the entire period of study.

Grateful thanks also to the non teaching stuffs of our department.

Lastly, the co-operation received from the classmates is also acknowledged.

ACKNOWLEDGEMENT