Biological Pathways Janick Mathys. Biological Pathways Definition Biochemical compounds Biological...

Biological Pathways

Janick Mathys

Biological Pathways

• Definition• Biochemical compounds• Biological interactions• Energy• Control interactions• Levels of abstraction• Types of biological pathways• Integration of pathways• Inference Issues

Biological Pathways

Definition:

A biological pathway is a sequence of interactions between biochemical compounds aimed at the maintenance and control of the flow of information, energy and biochemical compounds in the cell and the ability of the cell to change its behaviour in response to stimuli.

Biological Pathways

Definition:

A biological pathway is a sequence of interactions between biochemical compounds aimed at the maintenance and control of the flow of information, energy and biochemical compounds in the cell.

Main types of compounds in the context of pathways:

- proteins and protein complexes

- (part of) genes

- metabolites

Biochemical compounds

• (part of) genes• proteins and protein complexes• metabolites

– Amino acids and peptides : A, C, F, G, H, S…– Carbohydrates (sugars)– Cell-structure components – Cofactors, prosthetic groups and electron carriers: vitamins– Fatty acids and lipids – Nucleotides and nucleic acids: A, C, T, G

– Monocarbon compounds: CO, CH4, CH3OH

– Essential elements: S, P, O2, Fe, radicals

– Aromatic compounds:

compounds with special stability and properties

due to a closed loop of electrons (ring structure)– …

Biochemical compoundsgenes - Fundamental physical and functional units of heredity

- Ordered sequences of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product (i.e. protein or RNA molecule)

regulatory DNA sequences- Small conserved sequences that interact with special types of proteins (TF) thereby activating or repressing the expression of target genes

- Located in the promotor region in front of the target gene

geneRBSRegulatory sequences

Promoter

Biochemical compoundsproteins and protein complexesRas protein

Transcription initiation complex in eukaryotes

Protein: 3D-chain of amino acids that is represented as a linear sequence

of amino acid letter codes and performs a molecular function

Biochemical compoundsmetabolitesAny product of metabolism such as an intermediate or an end product that is excreted

Examples:

- amino acids e.g. cysteine

- carbohydrates e.g. glucose

- …

Biological Pathways

Definition:

A biological pathway is a sequence of interactions between biochemical compounds aimed at the maintenance and control of the flow of information, energy and biochemical compounds in the cell.

Biological Interactions:

SubstratesInteraction

Products

Energy

Biological Interactions

Main types of interactions in the context of pathways:

- Expression

- Assembly/Disassembly

- Transport

- Chemical reactions

SubstratesInteraction

Products

Energy


I. Expression:

The process by which a gene's information is converted into a protein Expressed genes are transcribed into mRNA and translated into protein or transcribed into RNA but not translated (transfer and ribosomal RNAs).

II. Assembly:

The formation of a complex of proteins, RNA and/or DNA with a molecular function that cannot be performed by the individual compounds

GeneExpression

Protein

Protein AAssembly

Complex

Protein B


- Expression: fus1 Expression Fus1

Cell fusion protein 1

DNANucleus

mRNA

fus1 DNA

fus1mRNA

AA1 AA2 AA3Fus1 protein

Yeast

- Assembly of complexes: Ribosomes:

- complexes of RNA and proteins

- translate genetic information into protein

Prokaryotes Eukaryotes


Detail: Assembly of the small ribosomal subunit in prokaryotes

Prokaryotes



Prokaryotes

ribosome

23S rRNA5S rRNA31 proteins

16S rRNA21 proteins


III. Transport:

Change of location of compounds

IV. Chemical reaction:

Compound A at location 1

Transport

Compound Aat location 2

Compound AReaction

Compound B

- Transport: a. Transport of nascent proteins through plasma membrane of the ER:

b. Transport of glucose from the lumen of the intestine into the blood:


Ribosome - nascent protein complex

in cytoplasm TransportNascent protein

in lumen of ER

Glucose

in intestineTransport Glucose

in epithelial cellTransport Glucose

in blood


a. Transport of nascent proteins into the lumen of the endoplasmic reticulum in eukaryotes

Plasma membrane of the ER

Eukaryotes

ER : organelle of eukaryotic cells consisting of a ± continuous system of membrane-bound cavities throughout the cytoplasm of a cell. Itsfunction is the transport of proteinsthat have to be secreted to the membraneof the cell.


b. Glucose transport from the lumen of the intestine into the blood stream


Higher Eukaryotes

- Chemical reactions:1. Redox reactions: Oxidation - Reduction (Photosynthesis):

transfer of e- from electron donors to electron acceptors

2. Phosphorylation - Dephosphorylation (Signal transduction):

addition/removal of phosphate groups

3. Hydrolysis:

breakdown of bonds in compounds through the addition of water

4. Splitting or forming of a C-C bond

5. Isomerisation:

Change of geometry or structure of a compound

6. Polymerisation

7. …


1. Oxidation – Reduction of NADH – NAD+:

Chemical Reactions

H+ + 2e- +

2. Phosphorylation:Phosphorylation cascade involved

in the uptake of glucose into the cell

Chemical Reactions

1. Non ionic glucose is pumped through the cell membrane, which is negatively charged2. A cascade of phosphorylations and dephos-phorylations takes place resulting in the phosphorylation of glucose as it enters the cell3. The ionic nature of Glucose-6-P prevents it from escaping back through the membrane

ATP ADP Prokaryotes

-OH

3. Hydrolysis:hydrolysis of lactose into

galactose and glucose by

beta-galactosidase

Chemical Reactions

1. Lactose is pumped through the cell membrane2. Hydrolysis of lactose into galactose and glucoseimmediately as it enters the cell => Extra step (energy cost) as compared with themetabolism of glucose

Prokaryotes

Fructose-6-P

4. Splitting C-C bonds: Cleavage of fructose-1,6-PP to dihydroxyacetone-P + glyceraldehyde-3-P

P has the size of fructose core => 2 negative P in close proximity => stress

5. Isomerisation:Rearrangment of Glucose-6-P into Fructose-6-P, a more compact and lower entropy (more unstable thus more willing to react) molecule

Chemical Reactions

Glucose-6-P

Phosphohexose isomerase

Fructose-1,6-PP DHAP + GA-3-P

Fructose-1,6-PP aldolase


• Energy is always required to form chemical bonds • Energy is sometimes released by the breaking of chemical bonds• For biological interactions the cell uses 3 energy sources:

- ATP: Adenosine TriPhosphate- GTP: Guanine TriPhosphate- Creatine phosphate

• ATP is generated by electron transfer in mitochondria:- Electron carriers pick up H+ and e- released by the breakdown of nutrients (e.g. glucose) - Electron carriers transfer H+ and e- to electron carriers in the mitochondrial membrane

- Transfer of e- through the mitochondrial membrane down to O2 releases energy - This energy is used to transport the H+ across the mitochondrial membrane- Rush of H+ releases energy used for phosphorylation of ADP to generate ATP

SubstrateInteraction

Product

Energy

Energy sources

• ATP: Adenosine TriPhosphate- Primary energy source of cells- Building block for DNA- High energy bonds between phosphates- Dephosphorylation of outer phosphate to form ADP releases 7.3 kcal/mol

• GTP: Guanine TriPhosphate- Secondary energy source of cells- Building block for DNA- Bound by G-proteins for signal transduction- High energy bonds between phosphates- Dephosphorylation of outer phosphate to form GDP releases 7.5 kcal/mol- (Cleaving the phosphate-ribose bond would release only 5 kcal/mol)

• Creatine phosphate- Extra energy source for muscle cells- Dephosphorylation releases 10.3 kcal/mol

! all nucleotides are full of energye.g. transcription: energy comes

from dNTPs themselves

Energy

Prokaryotes

ribosome

23S rRNA5S rRNA31 proteins

16S rRNA21 proteins

GTP provides energyfor the assembly of thelarge subunit and the 30S complex

Energy

Transport of nascent proteins

into the lumen of the ER in eukaryotes


Eukaryotes

GTP provides energy for bindingof ribosome to ribophorin and for insertion of peptide in the membrane

Energy

Glucose transport from the lumen of the intestine into the blood stream

ATP provides energy for transport of Na+/K+ out/in cell (against concentration gradient)

! Transport of glucose down concentration gradient: no ATP required


Higher Eukaryotes

Na+

Na+

Na+

Electron carriers

• NADH: Nicotinamide Adenine Dinucleotide- Oxidation to NAD+ releases 52.6 kcal/mol- Due to some inefficiency this only allows 3 ATPs to be formed

• FADH2: Flavine Adenine Dinucleotide- Oxidation to FAD releases 43.4 kcal/mol- Due to some inefficiency this only allows 2 ATPs to be formed

1. Oxidation – Reduction of NADH – NAD+:

Electron carriers

H+ + 2e- +

Control Interactions:

Enhance or repress other interactions

Main types of control interactions:

- transport facilitation

- enzymatic catalysis

- inhibition

- activation of gene expression

- repression of gene expression


+ -

Compound A at location 1Transport

Compound A at location 2

Facilitation

Transporter protein

+

I. Transport Facilitation:

Control Interactions


- Transport Facilitation:a. Facilitation of the transport of

nascent proteins into the lumen

of the endoplasmic reticulum by

ribophorin


Eukaryotes


Transport Facilitation:b. Glucose transport from the intestine into the blood stream is facilitated by

- Na+-glucose cotransporter pore complex

- glucose transporter protein

http://bio.winona.msus.edu/berg/ANIMTNS/FacDiff.htm

Removal of Na+ in epithelial cells is facilitated by Na+/K+ pump


Higher Eukaryotes

http://bio.winona.msus.edu/berg/ANIMTNS/FacDiff.htm


c. Facilitation of the transport of glucose and lactose into the cell by EIICB and lactose permease

Prokaryotes


II. Catalysis of chemical reactions by enzymes:

Enzymes:

Proteins (RNAs) that act as biological catalysts, speeding up reaction rate by reducing the amount of required energy

* by concentrating different substrates

* by inducing conformational changes in substrates through binding

Enzymes DO NOT participate in the reaction or alter its direction/nature

Compound AReaction

Compound B

Catalysis

Enzyme

+

- Catalytic Enzymes :1. Redox reactions: oxidase, dehydrogenase

transfer of e- from electron donors to electron acceptors

2. Phosphorylation - Dephosphorylation: kinase, phosphatase

addition - removal of phosphate groups

3. Hydrolysis: hydrolase

breakdown of bonds through the addition (- removal) of water

4. Transfer of a side group: transferases

5. Splitting or forming a C-C bond: desmolase, aldolase

6. Changing geometry or structure of a compound: isomerase, gyrase

7. Joining two compounds through hydrolysis of ATP: ligase

8. Polymerisation: polymerase



III. Inhibition:

Compound AInteraction

Compound B

Inhibition

Compound

-

Control interactions form a means of using compounds to introduce feedback !


- Inhibition

Lactoseextracellular

Transport

Lactoseintracellular

Facilitation

Lactose permease

+

-Inhibition

EIIAGlc

Inhibition of the transport of lactose into the cell in prokaryotesby a component (EIIAGlc) of theglucose transporter complex=> Catabolite repression

1. Glucose is pumped through the cell membrane2. A cascade of phosphorylations and dephos-phorylations takes place resulting in - the phosphorylation of glucose - the dephosphorylation of EIIAGlc-P into EIIAGlc

3. EIIAGlc shuts down the lactose permease, pre-venting lactose from entering the cell

Conclusion: Catabolite repression:This system ensures that bacteria give preference to the most energetic nutrient


IV. Activation of gene expression:

V. Repression of gene expression:

GeneExpression

Protein

Activation

Transcription factor

+

GeneExpression

Protein

repression

Transcription factor

-


Activation of gene expression:

- DNA is packaged into nucleosomes and higher-order chromatin structures

-Transcription factor binds specific regulatory element

- Transcription factor recruits chromatin remodeling and modifying complexes

- Transcription factor recruits components of the transcription initiation complex

- Transcription factor stimulates activity of assembled transcription complex


Activation of gene expression:


Transcription factor (complexes):• Proteins that bind to specific regulatory sequences in the DNA• Regulate the level of expression of target gene(s) by controlling whether

and how vigorously the gene is transcribed into RNA• The on/off switches and rheostats of a (group of) target gene(s)

Regulatory DNA sequences• Every gene has its own cis-acting regulatory sequences• Vary greatly in complexity among genes and organisms

When active transcription factors associate with the regulatory sequences of their target genes, they can function to repress (down-regulate) or induce (up-regulate) transcription of the corresponding RNA

Cysteine

Amino AcidsProtein

(Molecular Function)

Pathway(Biological process)

Biological Pathways

• Metabolic pathways• Developmental pathways• Signal-transduction pathways• Genetic regulatory circuits = genetic networks

• Pathways interact• Pathways overlap

=> Biochemical compounds are involved in different pathways

Metabolic Pathways

• Metabolism:The sum of all chemical reactions that take place within a cell providing energy for vital processes and for synthesizing new organic material

• EcoCyc/HinCyc/MetaCyc: Encyclopedia of Escherichia coli Genes and Metabolism

Encyclopedia of Haemophilus influenzae Genes and Metabolism

• EMP:Enzymes and Metabolic Pathways database

• KEGG: Kyoto Encyclopedia of Genes and Genomes

• …

Metabolic Pathways

• Biosynthesis = Anabolism: Sequences of enzyme-catalyzed chemical reactions by which complex molecules are formed in living cells from building blocks with simple structures

• Degradation = Catabolism:Sequences of enzyme-catalyzed chemical reactions by which large molecules in living cells are broken down or degraded into building blocks

• Transport:Sequences of transport (facilitation) interactions by which compounds are transported from one location to another.

• Energy Metabolism:Sequences of enzyme-catalyzed chemical reactions by which chemical energy obtained from the environment by degradation of nutrients or by capturing solar energy (plants) is transformed into energy-rich compounds that are required for metabolic processes

Metabolic Pathways

• Metabolism of:– Amino acids, peptides, proteins and derivatives– Carbohydrates (sugars)– Cell-structure components – Cofactors, prosthetic groups and electron carriers– Fatty acids and lipids – Nucleotides and nucleic acids – Monocarbon compounds – Essential elements – Aromatic compounds

Metabolic Pathways

• Glycolysis (Embden-Meyerhoff-Parnas pathway) : Degradation of glucose

to pyruvate for generation

of energy

Phases:

1. Preparatory phase:

- activation of glucose by

phosphorylations

- conversion to glyceraldehyde-3-P

by hexose splitting

Metabolic Pathways

• Glycolysis (Embden-Meyerhoff-Parnas pathway) :

Phases:

2. Pay-off phase:

- oxidation of glyceraldehyde-3-P

to pyruvate

- coupled formation of ATP and

NADH

Metabolic Pathways

Overlap and integration

of metabolic pathways:Amino acid metabolism of E.coli

Metabolic Pathways

Overlap and integration

of metabolic pathways:Entire metabolism of E.coli

Signal Transduction Pathways

Signal transduction

Genetic network

Phosphorylations!


• Mating reaction in yeast:

Two mating types (a – α)

- opposite types communicate by

secreting a pheromone

(a-factor – α-factor)

- exposure to pheromones causes

the cells to stop dividing, alter their

cell polarity and eventually to fuse

Yeast

Kinase Ste20 P

MAPKKSte3

MAPKKSte7MAPKK

Ste20

MAPKKSte5 PP

P

P

Nucleus

PSTOP cell cycle in G1

G protein

Inactive cell cycle arrest factor

Activated

Kinase complex

PSte12

fus1 gene

PInactive transcription factor Activated

Far1


• Overlap and integration of signal transduction pathways

Yeast Pheromone Signaling Pathway

Humans : MAPK signaling pathway : growth control -> tumor developmenthttp://www.bio.davidson.edu/courses/Immunology/Flash/MAPK.html

http://www.bio.davidson.edu/courses/Immunology/Flash/MAPK.html


• CSNDB:Cell Signaling Networks Database

http://geo.nihs.go.jp/csndb/

Contains information on biological compounds, their sequences, structures, functions and interactions which transfer cellular signals in human

Directed graph representation

Human MAPK signaling pathway

http://geo.nihs.go.jp/csndb/

Genetic networks

Signal transduction

Genetic network

Genetic networks

• Biochemical computers controlling the on/off switches and rheostats of a cell at the gene level

• Dynamically orchestrate the expression level for each gene in the genome by controlling whether and how vigorously that gene is transcribed

• Essential interacting components:

- Activated transcription factor complex

- Regulatory DNA sequences • Output : RNA and proteins• Some of these proteins are the actuators of inhibition and repression

=> main feedback loops• Co-regulated target genes often code for proteins that act together to build

a specific cell structure or to effect a concerted change in cell function• Often multiple waves of regulation with first wave products regulating

expression of another group of genes and so on

Genetic networks

Genetic networks• Genetic network controlled

by the CtrA response regulator

in bacteria

Nature Reviews Molecular Cell Biology 3; 167-176 (2002)

Genetic networks

• BRITE : Biomolecular Relations in Information Transmission and Expression

http://www.genome.ad.jp/brite/

Contains information on signal transduction pathways and the genetic networks they activate (genes, the control of their expression and proteins)

Directed graph representation

Still under construction

http://www.genome.ad.jp/brite/

Integration of Pathways

The integrated network of biological pathways is a cellular input-output device

1. Input signals are captured by appropriate receptors and transduced by signal transduction pathways

2. Signal transduction pathways activate genetic networks, causing changes in cellular protein and RNA composition

3. The changes in enzyme/inhibitor composition alter the activity of metabolic pathways resulting in changed cell behaviour, function and/or structure

Input Signals

Signal transduction

pathways

Genetic networks

Primary Outputs:

Changed RNA and protein composition

Metabolic pathways

Terminal Outputs:

Changed cellbehaviour and

structure

Feedback circuitry

Biological Pathways

• Inference Issues:

- Complete network of integrated biological pathways is blueprint of life

- Genome is only an archiving system of building blocks

- Regulatory DNA motifs are bar codes to retrieve the building blocks

• Combination of high-throughput experiments, prior knowledge and bayesian network inference is necessary

Experiments:

- Microarrays

- Proteome analysis

- Yeast two-hybrid

- Phosphorylations

Expression is result of underlying network

Biological Pathways

• First inference results

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