# Presentation: 090149

## We are working in Tier I with mechanisms

**Are mechanisms similar or**
**dissimilar across phyla, species,**
**classes, etc.?**

**In other words – **
**What is the phylogeny of**
**the various mechanisms?**

**Attempt at ‘Problem Statement’**

## In particular, what is the phylogeny of:

**Nuclear receptors**** – as they bind the**** the ligands and activate particular**** genes and pathways**** **
**P450s**** – as they synthesize and**** metabolize most NR ligands**

**Regrettably, will emphasis more of what**
**we don’t know, than what we know**

## Nuclear Receptors

**Metazoan signaling pathways**
**5 Basic evolutionary groups:**
** ****- based on DNA element structure****    (a repeated sequence, some ****      serial repeats, some mirrored,****      and with n nucleotides spacer)**
**- based on gene structure – intron/extron****     point in DNA binding region of protein**
**- based on amino acid sequence homology**

**At this time, steroid family (E, A, G, P, M) appears**
**to be unique to vertebrates.**

## Metazoans

**Hemichordates **
**Urochordates**
**Cephalochordates**** **
**Vertebrates**

**Gastrotrichs**
**Nematodes**
**Priapulids**
**Kinorhynchs**
**Onychophorans**
**Tardigrades**
**Arthropods**

**Bryozoans**
**Entoprocts**
**Platyhelminthes**
**Pogonophorans**
**Brachiopods**
**Phoronids**
**Nemerteans**
**Annelids**
**Echiurans**
**Molluscs**
**Sipunculans**
**Gnathostomulids**
**Rotifers**

**Metazoans**

***Bilateria***

***Deuterostomes***

***Protostomes***

***Lophotrochozoans***

***Ecdysozoans***

## Metazoans & Nuclear Receptors

**Nematodes**
**Priapulids**
**Kinorhynchs**
**Onychophorans**
**Tardigrades**
**Arthropods**

**Bryozoans**
**Entoprocts**
**Platyhelminthes**
**Pogonophorans**
**Brachiopods**
**Phoronids**
**Nemerteans**
**Annelids**
**Echiurans**
**Molluscs**
**Sipunculans**
**Gnathostomulids**
**Rotifers**

**Metazoans & Nuclear Receptors**

- ER, AR,
- GR, MR, &
- PR

- TR, ERR,
- VDR, RAR,
- PPAR

- Note: Greek letters indicate separate
- (multiple) forms of the receptor exist -
- action, ligand specificity, cell distribution
- may vary

***Vertebrates***
***only:***
***Steroid***
***Family (??)***

***“******Others”***
***also restricted***
***to a few phyla***

**Echinoderms **
**Hemichordates **
**Urochordates**
**Cephalochordates**** **
**Vertebrates**

## Implications

**1.  Is there a scientific basis or rationale****      to extrapolate an EAT based screen****      to other phyla?****At this time, probably not!****2. This means we have no screens for the****      other phyla.****They are unprotected!**

**We have serious scientific gaps that**
**need to be filled!!!**

## Metazoans & Nuclear Receptors

**Gastrotrichs**
**Nematodes**
**Priapulids**
**Kinorhynchs**
**Onychophorans**
**Tardigrades**
**Arthropods**

**Bryozoans**
**Entoprocts**
**Platyhelminthes**
**Pogonophorans**
**Brachiopods**
**Phoronids**
**Nemerteans**
**Annelids**
**Echiurans**
**Molluscs**
**Sipunculans**
**Gnathostomulids**
**Rotifers**

**??   ??    ??             RXR********COUP******

**EcdR, KNIRPS******

**For invertebrates, outside Arthropods**
**far less known about NucRec’s and **
**their transcriptional roles – some **
**are identified only as genes**

**Echinoderms **
**Hemichordates **
**Urochordates**
**Cephalochordates**** **
**Vertebrates**

## Briefly, the P450s

**Far more ancient**
**Far more diverse**
**But a parallel way to analyze**
**  ****the problem**

## Lipid synthetic

**Lipid synthetic**
**tree of ligands**

**DNA element**
**Several core**
**structures**
**intron-exon**
**break in**
**DNA region**
**organization**
**abcd  abcd**
**abcd dcba**
**nucleotide(s)**
**spacing**

## Suggested analysis

**What are the nuclear receptors that****  are potential ED targets?**
**What are the ligands (‘mimic targets’)?**
**What are the synthetic steps (P450scc,**
**  ****aromatase, 5-alpha reductase, ...)?**
**What then is the appropriate phyla for**
**  ****which a screen can and cannot be used?**
**  ****(And have a rationale basis to validate)**

## Nuclear Hormone

**DBD –**
**DNA Binding Domain**
**Zinc finger interaction**
**Often repeated**
**Several configurations**
**    ****distinguish receptor**
**    ****subclasses    **

**LBD –**
**Ligand Binding Domain**
**Large, 3 sided cavity**
**Envelopes ligand**
**Largely hydrophobic ****    amino acids lining**
**    ****the cavity**

**Nuclear Hormone**
**Receptor Structure**

## Nuclear Hormone

**Receptor Structure**

**AF-1**
**Activation Factor 1**
**(site for activation**
**by several kinase or**
**phosphorylation**
**pathways)**

**AF-2**
**Activation Factor 2**
**(interaction with other**
**protein families – one **
**represses and one activates)**
**Essential for transcription**

## Nuclear Hormone Receptor

**Nuclear Hormone Receptor**
**Ligand Triggering Action #1**

- NH – receptor
- Ligand Bound

**Heat Shock Protein**
**(HSP) chaperone**
**complex**

**CoRepressor**

**Dissociation**

**Receptor Dimer**
**Formation**

**This ‘free’**
**dimer appears**
**susceptible to**
**proteolysis**
**(ubiquitin)**

**Binding DNA**
**Element Sites**

**L**

## Transcribing Genes

**Into mRNA**

- RNA polymerase II
- or pol II complex
- actually transcribes,
- binds DNA at TATA box
- DNA site

- TATA Box                                               Gene Start (AUG codon)

**1. Pol II Binds**** TATA Box**

**2. Pol II Moves**** Down DNA**

**3. Pol II Initiates**** mRNA transcription**

- Pol II
- transcribing

- Base 0 for gene

- Base “X” # bases upstream for gene

## Nuclear Hormone Receptor-

**Ligand Complex Action #2**

- DNA Response Element                                                    TATA Box

- RNA polymerase II
- or pol II complex
- actually transcribes,
- starting at TATA box
- DNA site

** ****Response element for receptor****    is upstream of TATA box**** **
** ****RNA pol II normally cannot****     bind to TATA box alone,****     transcription blocked**

**Upstream of target**
** ****gene by X bases**

## Nuclear Hormone Receptor-

**Ligand Complex Action #2**

- DNA Response Element                                                    TATA Box

- RNA polymerase II
- or pol II complex
- must bind TATA box
- DNA site to transcribe
- target gene

**Receptor-ligand ****(*****holo*****-receptor) does**
**not interact directly**
**with the Pol II complex**

**Upstream of target**
** ****gene by X bases**

**Upstream of the TATA box Y bases**
**or X + Y of target gene**

## Nuclear Hormone Receptor-

**Ligand Complex Action 2**

**Recruitment of Coactivators is Necessary**
**They are an Obligate ‘Bridge’ to Pol II complex***

**** Receptor-ligand can bind DNA and NOT recruit pol II = Antagonist***

## Chemical Numbers vs Chemical ‘Doses’

***Working Examples***
***Perfume Raw Materials***

**Salvito et al.  *****Env. Toxicol. Chem.***** 21(6): 1301-1308**
**  ****Prediction of Environmental Concentrations (PECs)**
**  ****for > 2,100 chemicals**
**  ****Prediction of PNEC – PEC/PNEC ratio - prioritization**
**Simonich et al.  *****Env. Sci. Tech.***** 36(13): 2839-2847.**
**  ****Measurement and Validation of PECs**

## Thyroid Toxicological History

**Adverse Effects and Mechanisms**

**Thyroid – goiter/developmental toxicity**
**Mechanisms elucidated 1940-1990:**
**- Iodide deficiency or uptake blockade to thyroid**
**- Inhibition of thyroid peroxidase**
**- Blockage of thyroid T3/T4 release**
**- Increased T3/T4 metabolism****- Inhibition of 5’-deiodinase****- Life stage sensitivity vs consequences**
**Clear endpoints: thyroid histopathology, circulating****   T3/T4 and TSH**