Konsep Dasar Biokimia Sel dan Molekuler

Kusumo HariyadiDariSofia Mubarika (FK UGM)

NOBLE, D (2002) Nature Reviews Molecular Cell Biology 3, 460-463.Unravelling complexityNeed to work in an integrative way at all levels: organism organtissuecellular sub-cellularpathwaysprotein gene There are feed-downs as well as upward between all these levels Systems leveltriggers ofcell signalling Systems levelcontrols ofgene expression Protein machineryreads genes

Physiological systems & function Top-down

Middle-out

Bottom-up

Molecular data & mechanismssystemsorganstissuescellspathwaysorganellesSydney BrennerNobel Prize2001Middle-out!!

Mathematical Biosciences Institute (Ohio State Univ), 2 October 2003RNAProteinDNADOGMA CENTRAL

Central dogma of molecular biologyAdapted from http://www.bioinfbook.org/DNA Sequence(splited by genes)RNAphenotypeproteinAmino Acidsequence

3 Molecules of LifeAll life depends on three critical molecules: DNAsHold information on how cell worksRNAsAct to transfer short pieces of information to different parts of cellProvide templates to synthesize into proteinProteinsForm enzymes that send signals to other cells and regulate gene activityForm bodys major components (e.g. hair, skin, etc.)

DNAPhosphateBase (A,T, C or G)Sugar DNA is composed of basic molecules called nucleotides Each nucleotide contains phosphate, sugar and a nitrogen base. There are four bases: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)

DNA: structure The sugar-phosphate bonds of nucleotides form the backbone of a DNA strand. The four bases of DNA are arranged along the backbone in a particular order, which is called the DNA sequence. Two DNA strands are held together by weak bonds between the base pairs. Two possible base pairs: A-T, C-G. Two strands of DNA forms a double helix structure. Source of diagram: http://www.cs.utexas.edu/users/s2s/latest/dna1/src/page2.html

DNA: packaged in chromosomesEach chromosome is essentially a package for a very long, continuous DNA double strand.Lodish et al. Molecular Biology of the Cell (5th ed.). W.H. Freeman & Co., 2003.

DNA: splitted by genesgenepromoterExon 1Exon 2Exon 3Intron 1Intron 2 A gene is a region of DNA that carries the information for constructing a protein. 2-3% of humans DAN are genes, the rest is called junk DNA. Promoter is the upstream portion of a gene. It determines under which conditions the associated gene will be expressed. Most Eukaryotic genes have their coding sequences, called exons, interrupted by non-coding sequence called introns.

DNA: replicationsBefore a cell can divide, it must replicate all its DNA. From one end, the double helix is unwound, expose single bases on each strand. Each strand works as a template to reform a new double helix. The new double helix is formed by strict base pairing requirement, i.e., A-T, C-G.

RNASource of diagram: http://en.wikipedia.org/wiki/RNA RNA (ribonucleic acid) is the intermediary between DNA and protein. RNA is a single strand of nucleic acid. Instead of having nitrogen base T (Thymine), RNA has U (Uracil) Unlike DNA, which is located primarily in the nucleus, RNA can also be found in the cytoplasm. In nucleus, the gene code is transcribed to a RNA. Then RNA move out of nucleus into cytoplasm, where the RNA code is translated to amino acid sequence.

ProteinsProtein is a folded amino acid chain.20 different amino acids can be found in proteins.A proteins 3-D structure determine its functions in the cell.Proteins do all essential work for the cellbuild cellular structuresdigest nutrients execute metabolic functionsMediate information flow within a cell and among cellular communities.

Another view of central dogmaA gene is expressed in 3 steps:1) Transcription: RNA synthesis2) Splicing: removal of intron sequence from RNA3) Translation: Protein synthesis

Transcription Transcription is initiated by a complex of transcription factors binding to the promoter site located upstream of the gene

An enzyme, RNA polymerase II, travels along the gradually unzipped DNA template and polymerizes nucleotides into an RNA.

The sequence of nucleotides on DNA template determines the sequence on RNA by following the rule of base-pair complementarity, i.e., A U, T- A, C G, G C. Transcription continues until entire gene is copied to RNA.

Source of diagram: http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/chem/nucleic

Animation

Splicingpre mRNAExon 1Exon 2Exon 3Intron 1Intron 2mature mRNA

Translation (1) By translation, the nucleotide sequence on mRNAdetermines the amino acid sequence by genetic code. Genetic code: three base pairs of RNA (called a codon) determine one amino acid based on a fixed table. Translation always starts at AUG (start codon), and ends with any of UAA, UAG, or UGA (stop codon)

Translation (2)Transfer RNAs (tRNAs): small RNA molecules. Most of the tRNAs function as carriers of amino acids and participate in protein synthesis.

For example, the tRNA with the anticodon CGG corresponds with the codon GCC and attaches alanine amino acid onto the peptide chain.

Ribosome: a complex of protein and rRNA AnimationSource of diagram: http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/

SummaryCentral dogma of molecular biologyThree components DNARNAProteinThree steps transcriptionsplicingtranslation

More termsGenome: an organisms genetic material

Gene: a discrete units of hereditary information located on the chromosomes and consisting of DNA.

Genotype: The genetic makeup of an organism

Phenotype: the physical expressed traits of an organism

Nucleic acid: Biological molecules (RNA and DNA) that allow organisms to reproduce;

Modern Technology in MedicineHuman genome:Gene ther. in 2010Individualized Ther.New hopes:CancerVascular/Heart dis.DiabetesAlzheimersParkinsonModern technology:DiagnosticEarly ScreeningPrognosticFollow up Stem cell Antisense Biomedical EngineeringNew approaches:Genome Analysis:Expression gene analysisMicro ArrayProtein analysis

How much DNA do we have?humans have 2 x 23 chromosomesEACH cells contains 6 billion bases DNAthat is 1 meter of DNAa human being has >100.000.000.000.000 cellsthat is 100 billion km of DNA

Cell cell communicationReceptorsSignal transductionsSecond messengers moleculesNuclear transcription factorsGene activations Protein Respons

A process by which signals from outside the cell are transferred inside the cell through cascade of proteins interaction. Involved in all cellular activities (death, growth, differentiation, etc.)

Regulate by protein phosphorylation.

Cellular signal transduction

Proto-oncogenes encode components of growth factor signal transduction pathwaysComponents shown in yellow are known proto-oncogenes

The VEGF family and its receptorsAdapted from Ferrara N. Nat Med 2003;9:66976Migration, permeability, DNA synthesis, survivalLymphangiogenesis P PP P P PP P P PP P VEGF-A VEGF-B PlGFVEGF receptor-1VEGF-AVEGF receptor-2VEGF-C VEGF-DVEGF receptor-3Angiogenesis

VEGF signal transduction and its effectsShibuya M. Cell Struct Funct 2001;26:2535VEGF binding to VEGF receptor-2 activates a signalling cascade resulting in cellular effectsCation channel Permeability P PP P P PP P P PP P VEGFVEGF receptor-1VEGFVEGF receptor-2VEGF-CVEGF-DVEGF receptor-3DAGDAGPLCPLCProtein kinase CRaf-1MAPKProliferation, migration PermeabilitySAPK/ JNKApoptosisSurvivalCalcium releaseCa2+ProliferationMigrationIP3PLCP13KProtein kinase B

Agents targeting theVEGF pathwayVEGFVEGF receptor-2Cation channel PermeabilityAntibodies inhibiting VEGF (e.g. bevacizumab)Antibodies inhibiting VEGF receptorsSoluble VEGF receptors(VEGF-TRAP)Small-molecules inhibiting VEGF receptors (TKIs) (e.g. PTK-787)Ribozymes(Angiozyme) P PP P P PP P P PP P Migration, permeability, DNA synthesis, survivalLymphangiogenesisAngiogenesis

Signal Transduction from Receptor to Nucleus Via RAS p21

Regulators : proliferation, differentiation, apoptosis, repairGenome :Genes : Cell cycleDifferentiatioApoptosisRepairMetabolismetc

ProteomeProtein: cyclin, CDK,CDKIGF : GM-CSF, FGFBcl-2, p53, caspaseGadd, enzym repairdllTranscriptome

RNARNARNARNARNARNA

How to understood ??GENE:CytogeneticsFISHDNA analysisSouthern blot +/-PCR specific MLPASequencing : SSCP

Gene activ: RNANorthern blotRT- PCRRNA-se protection AssayMicro arrayR-ISH

Gene active : ProteinWestern BlotSDS-PAGEELISAFISH

27Im betting that an intimate understanding of cell cycle regulation and apoptosis is key to understanding the process of carcinogenesis.If nothing else, the main idea emphasized here is that the coupling between the cell cycle and apoptosis how these processes are coordinated is key.This talk will focus on the links and controls of the onset of the cell cycle and apoptosis.Two types of VEGF receptors have been identified. VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR/Flk-1) bind VEGF with high affinity. Both VEGF receptor-1 and VEGF receptor-2 have seven immunoglobulin (Ig)-like domains in the extracellular domain (ECD). VEGF receptor-2 has the highest binding affinity for VEGF165.1Another member of the receptor family with seven Ig-like domains in the ECD is VEGF receptor-3 (Flt-4). Though not a receptor for VEGF, it binds VEGF-C and -D. In the embryo, VEGF receptor-3 is expressed in angioblasts and venules. Later on in development, it becomes restricted to lymphatic endothelium, suggesting that it has a role in the regulation of lymphangiogenesis.1

Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:66976. Evidence suggests that VEGF receptor-1 and VEGF receptor-2 have differing signal transduction properties and may mediate different functions.VEGF receptor-2 undergoes strong ligand-dependent tyrosine phosphorylation in intact endothelial cells and mediates mitogenesis and chemotaxis in response to VEGF, whereas VEGF receptor-1 produces weak or undetectable responses. It has been suggested that VEGF receptor-1 is not a signalling receptor but a decoy receptor, negatively regulating the activity of VEGF on the vascular endothelium by sequestering and rendering this ligand less available to VEGF receptor-2. Conversely, later studies show that VEGF receptor-1 can interact with signal-transducing proteins and generate a mitogenic signal. More recent studies suggest that VEGF receptor-1s main role is as a ligand-binding receptor, rather than a signal-transducing receptor.1VEGF is also thought to have a role in the control of HSC survival and repopulation by means of an internal autocrine loop, i.e. the ligand interacts with its receptors within the cell.2 Mouse studies suggest, in contrast to their action on endothelial cells, that both VEGF receptor-1 and VEGF receptor-2 have roles in HSC development and survival.2 These observations could have implications for the toxicity of intracellular receptors blockers, e.g. tyrosine kinase inhibitors such as PTK787, which may interfere with the autocrine loop. Externally activating agents such as bevacizumab would not be expected to have these effects.Stimulation of signalling through VEGF receptor-2 on endothelial cells by VEGF binding results in the activation of a number of signalling pathways, including the MAPK and ras pathways. This results in cellular effects including endothelial cell proliferation, survival and migration, as well as increased vascular permeability.3

Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 1999;56:794814.Gerber H-P, Ferrara N. The role of VEGF in normal and neoplastic hematopoiesis. J Mol Med 2003;81:2031.Shibuya M. Structure and function of VEGF/VEGF-receptor system involved in angiogenesis. Cell Struct Funct 2001;26:2535. Over expression of VEGF by tumour cells can be targeted by:antibodies against VEGFantibodies against VEGF receptorssoluble VEGF receptors that bind circulating VEGFsmall molecule inhibitors of VEGF receptorscatalytic RNA molecules (ribozymes), which cleave VEGF receptor mRNA.