Biochemistry Flashcards
Essay Details
1000 word essays –
This is the maximum
Excluded from word limit – title, headings, sub-headings, legends, figures, tables,
reference list and in text citations
Follow the formatting guidelines – if you do not follow them, your mark will be capped
at 65% - the grade descriptors refer to professional norms
Minimum 1.5 line spacing
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Harvard referencing only! ‘et al’ should be in italics with a full stop then a comma.
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Include the word count in your submission
REMEMBER TO DO THIS
*You will also need to produce an annotated bibliography for 5 of your references – no
more than 2 review articles, the rest should be primary research.
Tips for essay:
- Focus on one transcription factor and one disease > can introduce nuclear receptors / TF in the intro.
- Discuss > give arguments for and against, consider the implications of. Don’t need to go into depth in the biochem structure etc. Do NOT include too much of the structural biochem, focus on the clinical / disease implications and pharmacology!
- Intro > give an overview of the essay, mention all the other diseases and processes ChREBP is involved in but then say what you are focusing on! Can use sub headings in the body. ‘Mechanism action of ChREBP in Metabolic Pathways’
Conclusion > do NOT add any new knowledge. - Submit 1 page of coursework for feedback and can book and 1-1 session.
- Make sure you fulfil all the formatting guidelines so the mark is not capped!
How to get a good mark:
- Analyse and discuss the info > discuss! You need a good flow, so what? Why does it matter? Now what, what does it lead into? Every sentence must have a purpose > key information! Use words such as however, as a result, because of this, according to this source, this evidence suggests…
- Merge references, studies show that… however this study showed the opposite…
- Integrate detail, DO NOT just relay information, critically analyse!
- Refer to figures in the text!
-Conclusion > therapeutic relevance, metabolic implications, biomarker use to treat cancer?
Overview of nuclear receptors
Nuclear receptors: DIRECT activation by ligands:
e.g. PPAR, PPAR/, PPAR
Nuclear receptors: INDIRECT activation by ligands:
e.g. SREBP
CELL COMMUNICATION
There are four basic mechanisms for
cellular communication:
a. Contact-dependent (direct contact) - Molecules on the surface of one cell are recognized by receptors on the adjacent cell, lock and key
b. Paracrine signalling - Signal released from a cell has
an effect on neighbouring cells
c. Synaptic signalling - Nerve cells release
neurotransmitter which binds to
receptors on nearby cells
d. Endocrine signalling - hormones released from a cell
affect other cells throughout the
body
CELL BEHAVIOUR
Depends on multiple extracellular
signal molecules
* Each cell displays a set of
receptors allowing it be stimulated
by a set of ligands produced by
other cells
* These molecules work in synergy
to regulate the cell’s behaviour
Different cell types - Will respond differently to the
same extracellular signal
molecule.
SIGNAL TRANSDUCTION
Protein kinases and phosphatases
used in most signalling pathways
- Addition of phosphate group to polar group of
amino acid - Changes protein from hydrophobic to
hydrophilic polar - Enables protein to alter conformation when
interacting with other molecules - Phosphorylated amino acids able to interact with
other proteins - Allows proteins to easily assemble and
dissemble protein complexes - First steps involved in coordinating many cellular
functions - proliferation, metabolism, apoptosis,
inflammation and subcellular trafficking
GTP BINDING PROTEINS
Often used in signal transduction as
on/off switches
SECOND MESSENGERS - amplification of extracellular signal
cAMP
cGMP
DAG
IP3
RECEPTOR TYPES
ION-CHANNEL COUPLED RECEPTORS
In the presence of ions = opening o the channel. Channel opens or closes in response to concentration of signal ligand or membrane potential.
e.g. GABA –A and nicotinic ACh
receptors
G PROTEIN COUPLED RECEPTORS (GPCR)
Largest group >700 GPCR
Respond to neurotansmitters and hormones
Main target for drugs
External ligand binding to receptor activates an intracellular GTP-binding protein which regulates an enzyme that generates an intracellular second messenger.
ENZYME COUPLED RECEPTOR
signal molecule binds = active catalytic domain
E.g. Tyrosine Kinase
Ligand binding activates tyrosine kinase activity by autophosphorylation. Kinase activates transcription factor, altering gene expression (kinase cascade).
NUCLEAR RECEPTOR
Hormone binding allows the receptor to regulate the expression of specific genes.
General
features of
signal
transduction
Specificity – receptors only
on certain cell types
e.g. thyrotropin-releasing
hormone – response in
anterior pituitary not
hepatocytes – no receptors
Adr – alters glycogen
metabolism in hepatocytes
not adipocytes.
Receptors present but no
glycogen or metab enzyme in
adipocytes
HORMONES
Act via receptors in target
cells
High affinity – cells respond
to low [hormone]
Metabotropic receptors -
activate or inhibit enzyme
downstream from receptor
Ionotropic – open or close
ion channel in plasma
membrane – change in
membrane potential or ion
concentration
DIRECT REGULATION OF TRANSCRIPTION
BY HORMONES
- Hormone carried to the target tissue on serum binding proteins, diffuses across the plasma membrane and binds to its specific receptor protein in the nucleus.
- Hormone binding changes the conformation of the receptor. It forms homo or hetero dimers with other hormone receptor complexes and binds to specific regulatory regions called hormone response elements (HREs) in the DNA adjacent to specific genes.
- Receptor attracts Coactivator or corepressor proteins and with them regulates transcription of the adjacent genes, increasing or decreasing the rate of mRNA formation.
- Altered levels of the hormone regulated gene product produces the cellular response to the hormone!
NUCLEAR RECEPTORS and their impact on metabolism
48 identified to date in the human genome
➢ Homeostasis, reproduction, development and metabolism
➢ Metabolic receptors:
▪Energy/glucose metabolism (PPAR; PPAR)
▪Cholesterol transport/absorption (LXR)
▪Bile acids (FXR, LXR)
▪Xeno/endobiotics (PXR, CAR)
▪Cholesterol and fatty acid synthesis (SREBP)
➢Currently, targets for 10% of drugs in use
There’s a number of groups of nuclear receptors with similar structures = SUPERFAMILY > look up picture and examples.
ORPHAN RECEPTORS
Bind and are activated by currently unknown signalling
molecules (ligands, neurotransmitters, or hormones)
Share structural components with identified receptors whose signalling
molecules are already known
ALTERNATIVE CLASSIFICATION
Nuclear receptors are also classified according to their physiological ligands
and potential function
Adopted after the discovery of
their ligands.
Characterized by a lower
affinity for their ligands and
lower transcriptional activity.
Steroid hormone receptors, TR, VDR,
retinoic acid receptors (RARs)
characterized by high affinity for their
ligands and high transcriptional activity
Physiological ligands are
unknown, but may have
synthetic ligands. Often
functional inhibitors of
transcriptional activity of other
NR
look at slide 35 for essay!
NUCLEAR RECEPTORS:
DIRECT ACTIVATION BY LIGANDS
Examples - include members of subfamily 3 e.g. androgen receptor, oestrogen
receptors, glucocorticoid receptor, and progesterone receptor
n the absence of
ligand, located in the
cytosol
Hormone binding
triggers
* dissociation of heat
shock proteins
(HSP),
* Dimerization
* translocation to the
nucleus, where the
NR binds to
hormone response
element (HRE)
CLASS II NUCLEAR RECEPTORS
Examples - subfamily 1, e.g. retinoic acid receptor, retinoid X receptor and thyroid
hormone receptor
Retained in the nucleus
regardless of the ligand binding
status and bind as heterodimers (usually with RXR) to
DNA.
* often complexed
with corepressor proteins.
* Ligand binding to the nuclear
receptor causes dissociation of
corepressor and recruitment
of coactivator proteins.
* Additional proteins incl RNA
polymerase are then recruited
to the NR/DNA complex that
transcribe DNA into messenger
RNA.
INDIRECT ACTIVATION
SLIDES
NUCLEAR RECEPTORS: DOMAIN
STRUCTURE
(LIGAND GATED NR)
Ligand binding domain and DNA binding domain.
Hinge region = nuclear localisation signals.
DOMAIN A/B
Most variable – size and sequence
* Constitutive activating function (AF-1)
* Ligand-independent
* Phosphorylation site
DOMAIN C
▪ Most conserved domain
▪ DNA-binding domain:
* Targets receptor to specific DNA sequences (response
elements)
* 9 cysteines + other residues – high affinity binding
* Two Zinc fingers and COOH-terminal extension (CTE)
ZINC FINGERS
AAs required for discrimination of
core DNA recognition motifs in “P
box”
* Other residues in “D box” are
involved in dimerization
two α-helices:
1 -binds the major groove of DNA making
contacts with specific bases
2 - spans the COOH terminus of the
second zinc finger – at right angles
Steroid receptors > Ligand dependent binding. Sequence and spacing of core
recognition motifs determine
DNA binding specificity
DOMAIN D
Hinge region: DBD & LBD
▪Nuclear localization signals
▪ or
▪Domains for binding of co-repressors
DOMAIN E/F
Multi-functional:
1) Ligand binding
* AF-2: Ligand-dependent transcription activation
2) Mediates homo/hetero dimerization:
* Leucine zipper sequence
3) Protein-protein interactions
ChREBP
WHAT IS CHREBP?
* Carbohydrate signalling transcription factor – key metabolic regulator
* Name derived from interaction with carbohydrate response element sequences of
DNA
* Encoded by MLXIPL gene – chr 7
* encodes basic helix-loop-helix leucine zipper transcription factor of Myc/Max/Mad
superfamily - regulate cell proliferation, differentiation, and death
CHREBP IN TISSUES
ChREBP - regulates expression of specific genes coding for enzymes in glycolytic
and gluconeogenic pathways
Expressed in liver, white and brown adipose tissue, intestine, muscle, and
pancreatic β-cells - sites of active lipogenesis
Liver»_space; adipose > intestine > kidney = muscle (also in regions of brain)
* Induces de novo lipogenesis from glucose in response to glucose flux
into adipocytes
* Liver - glucose induction of ChREBP promotes glycolysis and lipogenesis
STRUCTURE OF CARBOHYDRATE RESPONSE ELEMENT
BINDING PROTEIN (CHREBP)
Look at pic on slides - week 1
Two isoforms – α&β
low glucose inhibitory domain
glucose activated conserved element
CHREBP - MECHANISM
* Coordinates synthesis of enzymes needed for carbohydrate and fat synthesis
* Inactive in phosphorylated form and located in cytosol
* When phosphoryl group removed from ChREBP by PP2A - transcription factor enters
the nucleus
* In nucleus
* nuclear PP2A removes another phosphoryl group,
* ChREBP joins with Mlx, and switches on synthesis of enzymes: pyruvate kinase;
fatty acid synthase; and acetyl-CoA carboxylase
CHREBP AND GLUCOSE METABOLISM
can direct glucose metabolism from
oxidative phosphorylation to
anabolic pathways
required for the proliferation of
various cell types
PENTOSE PHOSPHATE PATHWAY – REGULATION OF
CHREBP
High
glucose > Pentose
phosphate
pathway
PP2A - allosterically activated by xylulose 5-phosphate
PP2A > Dephos –Ser – ChREBP enters nucleus
Dephos – Thr – activates ChREBP - associates with Mlx
Serum glucose elevated – isoforms of Glut2 and kinase
(hexokinase IV) in hepatocytes -rapid uptake and equilibration
of intracellular glucose levels
Flux of glucose promotes formation of xylulose-5-phosphate
(Xu-5-P) - activates protein phosphatase 2A (PP2A)
dephosphorylate ChREBP
promotes nuclear localization and DNA binding
EVIDENCE FOR ROLE OF CHREBP
ChREBP suppression in HCT116 cells and HepG2 hepatoblastoma cells
- decreased aerobic glycolysis and anabolism
- decreased synthesis of lipids and nucleotides
ChREBP suppression
- reduces glucose-induced pancreatic β-cell proliferation
- reduces mRNA levels of cell cycle regulators
ChREBP over-expression
- promotes glucose-stimulated β-cell proliferation
CHREBP
AND
CANCER
Cancers - characterised by reprogrammed glucose
metabolisms - fuel cell growth and proliferation
DISTURBANCE OF METABOLISM AND TUMOUR
DEVELOPMENT
* Many human tumours - high rate of aerobic glycolysis, de novo fatty acid synthesis
and nucleotide biosynthesis
* ? increased glucose metabolism - lipogenesis & nucleotide biosynthesis –
enhanced tumour cell growth and proliferation
* Increased de novo fatty acid synthesis needed by cancer cells - construction of
lipid membranes
* Inhibition of fatty acid synthetase inhibits cell proliferation and increases
apoptosis of tumour cells
CHREBP AND COLON CANCER
Lei et al. Carbohydrate response element binding protein (ChREBP) correlates with colon cancer progression and
contributes to cell proliferation. Sci Rep 10, 4233 (2020). https://doi.org/10.1038/s41598-020-60903-9
ChREBP mRNA and protein expression - significantly increased in colon cancer tissue
compared to healthy colon, and expression was positively correlated to colon
malignancy
In vitro, ChREBP knockdown inhibited cell proliferation and induced cell cycle arrest
in colon cancer cell lines
Glycolytic and lipogenic pathways were inhibited but the p53 pathway
activated after ChREBP knockdown
Indicates - ChREBP expression is associated with colon malignancy and might
contribute to cell proliferation by anabolic pathways and inhibiting p53
?ChREBP as a useful biomarker to evaluate malignancy of colon cancer
ChREBP knockdown inhibition of colon cancer cell proliferation and induced cell cycle
arrest
TUMORIGENESIS
Glucose uptake, glycolysis and lactate production are increased in tumours &
proliferating/developing cells in the presence of sufficient oxygen and
mitochondrial function - “Warburg effect” / “aerobic glycolysis”
ChREBP required for proliferation of HCT116 colorectal cancer cells
ChREBP suppression in HCT116 cells - decreases aerobic glycolysis and anabolism,
decreased synthesis of lipids and RNA and reciprocally increased mitochondrial
oxygen consumption
ChREBP switches from oxidative phosphorylation to aerobic glycolysis in cancer cells
In breast cancer [ChREBP mRNA] correlates with tumour progression
ChREBP and Colon Cancer
https://www.sciencedirect.com/science/article/pii/S0925443916303234
(Use this for other reference ideas)
Include pictures from the lecture slides!
Add to the role of ChREBP in glycolysis, lipogenesis and gluconeogenesis using other references.
Add to the future therapeutic use of ChREBP.
ChREBP to be a transcription factor that binds to carbohydrate response element (ChoRE) in the promoter of pyruvate kinase, liver and RBC (Pklr)
[7]
. ChREBP is also termed “MLX interacting protein like” (MLXIPL) and Williams–Beuren syndrome chromosomal region 14. ChREBP (a basic-helix-loop-helix leucine zipper protein) leads to formation of a heterodimer with a Max-like protein (MLX)
[8]
,
[9]
,
[10]
. ChREBP is expressed in the liver, white adipose tissues (WATs), brown adipose tissues (BATs), the intestine, muscle, and pancreatic β-cells
[7]
,
[11]
. ChREBP regulates gene transcription in glycolysis, de novo lipogenesis, and other pathways
[11]
, which suggests that ChREBP has an important role in the pathogenesis of metabolic diseases and cancers.
ChREBP is a basic-helix-loop-helix leucine zipper transcription factor
[7]
,
[8]
. ChREBP and MLX form a complex. The ChREBP/MLX complex binds to the ChoRE of ChREBP target genes in the nucleus.
ChREBP has two isoforms: α and β (
Fig. 1
A)
[15]
. In health, ChREBPα is localized mainly in the cytosol. Upon glucose stimulation, ChREBPα is translocated from the cytosol to the nucleus. ChREBPα induces ChREBPβ transcription
ChREBPα has a low glucose inhibitory domain (LID) and a glucose response conserved element (GRACE)
[15]
,
[16]
. In contrast, ChREBPβ has only GRACE and lacks LID
[15]
. Only under low glucose conditions, LID can inhibit the ChREBP activity conferred by GRACE.
Nevertheless, this two-step activation mechanism seems to be important to reach the glucose threshold for ChREBP-mediated gene expression and makes ChREBP a “glucose switch”.
ChREBP is activated through metabolite signals derived from glucose
Glucose stimulates ChREBP transactivity through several metabolites derived from glucose. Xylulose-5-phosphate, glucose-6-phosphate, fructose-2.6-bisphosphate, UDP-GlcAc, and acetyl CoA are candidate “glucose signals”.
Xylulose-5-phosphase (Xu-5-P), glucose-6-phosphate (G-6-P), and fructose 2,6-bisphosphate are thought to activate ChREBP transactivity
[18]
,
[20]
,
[21]
,
[22]
,
[23]
,
[24]
,
[25]
,
[26]
. Xu-5-P and G-6-P were thought to activate ChREBP transactivity through de-phosphorylation and allosteric modification of ChREBP.
ChREBP is suppressed by “starvation” signals.
Upon starvation, plasma levels of glucose are decreased, whereas those of glucagon and epinephrine are increased. Free fatty acids (FFAs) are produced through lipolysis by glucagon and epinephrine (abbreviated in this figure) in adipocytes. FFAs are taken up in the liver, and levels of AMP and ketone bodies are increased. AMP and ketone bodies inhibit ChREBP transactivity through allosteric effects. AMP also inhibits ChREBP transactivity by AMP-activated protein kinase. Glucagon and epinephrine (abbreviated in this figure) also increase cAMP levels, resulting in ChREBP inhibition through cAMP-dependent protein kinase (PKA).
de novo lipogenesis
ChREBP and SREBP1c regulate common genes in the pentose phosphate shunt and lipogenic pathway. hREBP also induces Tkt expression, an enzyme that produces Xu-5-P
[44]
,
[48]
. Activation of Xu-5-P-activated protein phosphatase (PP2A) causes an increase in ChREBP activation and glycolytic flux through phosphofructokinase activation mediated by dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.
Glycolysis
ChREBP regulates expression of Glut2 and a glycolytic rate-limiting enzyme (Pklr).
Gluconeogenesis
ChREBP regulates G6pc expression > G6pc has an important role in gluconeogenesis. Insulin suppresses expression of G6pc. Other gluconeogenic genes (fructose-1,6-bisphosphatase, glucagon receptors) are also induced by ChREBP activation (
Table 1
), so carbohydrate-induced activation of ChREBP dominates over the suppressive effects of insulin to enhance glucose production.
CANCER
“The Warburg Effect” - EXPLAIN.
Therefore, the Warburg effect is, in one sense, efficient for cell proliferation. https://www.science.org/doi/epdf/10.1126/science.1160809?src=getftr
ChREBP is required for the proliferation of HCT116 colorectal cancer cells
[97]
. ChREBP suppression in HCT116 cells decreases aerobic glycolysis and anabolism, and is accompanied by decreased synthesis of lipids and RNA and reciprocally increased mitochondrial oxygen consumption
[97]
. Moreover, ChREBP inhibition leads to p53 activation and induction of arrest in HCT116 cells
[97]
. These findings suggest that ChREBP has a key role in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity, and that ChREBP switches from oxidative phosphorylation to aerobic glycolysis in cancer cells.
Further investigation is needed to clarify why the role of ChREBP in tumorigenesis might be dependent on different cell types.
https://www.sciencedirect.com/science/article/pii/S0925443916303234
Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucose-responsive regulation
https://pubmed.ncbi.nlm.nih.gov/11470916/
SREBP
(Sterol Regulatory Element Binding Proteins)
A key regulator of lipogenesis. It regulates a huge number of genes.
Transcription factor of basic helix-loophelix leucine zipper family.
Insulin modulates the SREBP pathway.
