Molecular الحلقة biology 14 واألخيرة To put you back in the context; the discussion was about Trancriptomics (the study of transcription). The following topic will be PROTEOMICS, which is the study of proteins, analysis of large sets of proteins. An important property of the field is the high throughput, which means that we're dealing with tens, hundreds, maybe thousands of specimens and genes Genomics Vs. Proteomics: Genomics are constant relatively: same genome for an individual along life (minor changes). Proteomics are dynamic: changes happen in min's, hours along the day (sleep/wake, eat/starve ). Regarding techniques of proteomics, there is: Slide#75 Mass spectrometry: An old method which went back in use, it measures mass/charge ratio. Consists of: -Ion source: Ionization of the specimen. -Mass analyzer: separation of ions based on size. -Detector: to detect if there is an ion or not, and where is it detected. Now every peptide has a distinct profile, that might not be enough so we need another go (in the first time we detect general properties of protein, maybe the protein family, so we need to specify this more).. Another ion source and mass analyzer are used, one of the proteins of the first go will be fragmented and separated in the analyzer, detected finally on the detector, and now we know which exact protein is this. An advantage of this method is that we can analyze a large number of proteins within one experiment AFTER being processed in liquid chromatography (this separates them based on size, polarity ). e.g. A blood sample is a complex sample that contains many proteins, we can identify all proteins by the aforementioned method. Extra note : in the figure, HPLC: highperformance liquid chromatography. CID: chemically induced dynamic sth Slide#77: (مصفوفة= array Protein microarray: (recall that Expression array Forwardphase Backwardphase Protein microarray Interaction array Functional array
-In forward phase, different probes (Antibodies usually) are on the array, distributed as spots. When the protein sample is administered on the array the antibodies will bind to their compatible proteins. So the goal of this (forwardphase) is to detect if the PROTEINS are present. Hundreds of proteins can be detected in the trial, each for its Ab. -In backward-phase, the goal is to find ONE or two proteins only, using one or two Ab and many specimens (hundreds). Forward One specimen Many probes Many proteins to detect(protein profile will result) Forward Vs. Backward: Backward Many specimens One or two probes One or two proteins to detect Slide#78: Interaction array: Human and fly have high gene homology, so where did human complexity come from? Not only expression! Modification, phosphorylation, glycosylation.. give role (protein-protein interactions). And this method is to detect these interactions. -The array contains known molecule ligands, let's say it contains proteins 1-100. Now when the specimen protein is added it bind and interacts with protein 2, 50 for say now I know that my protein reacts with 2 and 50. Slide#79: Functional array(aka enzymatic microarray): The goal is to know which substrate is for that specific enzyme, so the array contains enzymes, then the substrate is added, for example it reacted and gave products by enzyme #10 Enzyme #10 is the catalyst for this reaction. Expression Vs. Interaction Vs. Functional Expression Interaction Functional Does protein Does protein Does substrate exist? interact? react? Slide#80: Protein-Protein interaction: When studying a specific mutation (defective protein), we want to know what are the relationships and effects on other proteins in the cell. e.g. in the cell cylce (right figure), there are proteins A,B,C each one can regulate a point in the cycle, and can interact with other proteins. On the left figure (taken for multiple sclerosis disease) you can see the connections that make a network of interactions. So the purpose of studying protein-protein interaction is to predict the possibility of developing a disease if a gene was mutated. e.g. gene 1 can strongly affect genes 11,12,13 if mutated, while 13 can weakly affect 4,5,6.. just a network. Slide#81: The future of biomedicine is PERSONAL MEDICINE! :Giving the most suitable treatment depending on the expression profile of genes. Slide#83 The figure shows cancer steps, simple; tumor is at first a benign mass of cells that can be removed UNCONTROLLED
proliferation Accumulation of mutations one cell will become aggressive Proliferation cancer invasion and metastasis. Slide#84 Clonal= come from one cell origin. -Not all cells in tumor are homogenous, clusters of different clones can be found due to different mutations. Slide#85 -Hereditary 10% (Colon, eye, breast..) -As for estrogen, women with increased TIME or AMOUNT of estrogen have higher breast cancer susceptibility. Exogenously administered estrogen may help women at menopause and decrease osteoporosis, but must be given carefully. -Smoking is carcinogen -H. Pylori causes cancer by changing the environment of the stomach. -Liver cancer is #2 in Egypt! Due to high HCV infections. -#1 is Bilharsza, causing bladder cancer. Slide#86 & 87: Normal body cells have homeostasis, a balance between growth and death, #dying cells= #proliferating. If # proliferating cells or #dying cells tumors cancers. So on the balance; Oncogenes genes that increase cell proliferation. Tumor supressor genes increase genes that cell death and supress proliferation. Slide#89: Oncogene, when it is normal it is called proto-oncogene; normally it: Proliferation DIFFERENTIATION (because it leads to cell death faster) Cell survival BUT when mutated, the oncogene gives UNCONTROLLED: Proliferation DIFFERENTIATION Cell survival Slide#89 Extremely important (compare to tumor suppressor gene mutations in the next page) Oncogene mutations are DOMAINANT; one allele mutation is enough to cause cancer. The mechanism is GAIN of function; "Gain of function mutations change the gene product such that it gains a new and abnormal function. - Wikipedia" In the next 3 slides, examples of the 3 functions of oncogenes; cell proliferation, differentiation, and survival. Slide#90 Proliferation; The Ras gene: -Mutated in 95% of pancreatic cancers. -20% of other cancers. -Mechanism: Ras protein is activated by a receptor (e.g. epithelial growth factor receptor-egfr(erbb), or HER2) it attaches to the membrane from the inside (a key step) activation of a kinase (Raf) activation of another kinase (MEK)
activation of another kinase (ERK) ERK will do its job inside the nucleus; activating transcription factors, causing transcription of some genes, e.g. cyclind1 induce cell proliferation by inducing the cell cycle from G1 to S phase. CyclinD1 also phosphorylate Rb gene (retinoblastoma), inactivating it, allowing cell proliferation. Note: breast cancer can develop via Ras mutation activation of HER2 receptor gene amplification "A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes.- Wikipedia". The chromosome may look thicker in some bands. Suggested therapy: inhibition of the enzyme that helps Ras protein binding the membrane (blocking the key step aforementioned). It worked perfectly in labs on mice, but failed on clinical trials. Slide#91: Differentiation; Promylocyte(PML)/RARα gene. This gene when mutated blocks differentiation on a certain cell of the lineage which is the Promyelocyte thus leading to overproliferation of it Promyelocytic leukemia. Suggested therapy: drug to induce cell differentiation rather than proliferation. Slide#92: Survival(prevention of cell death); Akt: Akt is activated by a kinase (PIP3- Phosphatidyl inositol triphosphate) which is activated by a receptor (e.g. receptor A7) Akt activates Bcl-2 blocks mitochondrial induced apoptosis no apoptosis accumulation of mutations Cancer. Slide#93: Tumor suppressor genes inhibit proliferation and induce cell death. Type of mutation is RECESSIVE (Both alleles must be mutated). Loss of function mutation "are the result of gene product having less or no function.-wikipedia". -In children born with 1 Rb allele deleted, if the other is mutated this will cause loss of function eye cancer. Slide#94: How RB works? (see figure) - Sequestration binding of E2F (elongation factor 2) inhibiting G1 to S progression in the cell cycle. If Rb was phosphorylated inhibited cant bind E2F G1 to S will progress normal. (Recall that Rb gets phosporylated by cyclingd1 which expression is increased by the Ras pathway) Slide#95: P53 is a tumor suppressor gene, activated by DNA damage. -If the damage can be repaired cell cycle will be arrested repair will take place cell will return to its cycle. -If the damage was huge Appoptosis. -How P53 works? -It induces p21 (a protein) expression
p21 will inhibit 1)Cdk's (cyclin dependant kinase) inhibiting cell cycle. 2)PCNA (Proliferating cell nuclear antigen) inhibiting DNA replication. Slide#96: Genetic model of colorectal cancer: ordered sequential mutations resulting in metastatic cancer. Discovered by Bert Vogelstein, the model didn't work on all types of cancer. -One mutation isn t enough to make the cell cancerous, mutations must be accumulated. -Normal cell APC mutation dysplastic cell Ras mutation intermediate adenoma another mutation late adenoma another carcinoma another metastatic cancer. -Some companies are developing kits to decide if a certain case of colorectal cancer has a Ras mutation or not, providing treatment upon result (personalized medicine). Slide#97: In traditional treatment of cancer, many cases will not completely be cured, some cancerous cells might survive because they have different mutation cancer may reappear. 2)HER2 amplification give HERCEPTIN (a drug). i.e. give the drug for the specific mutation according to patient profile. The slide shows molecular diagnosis of cancer. Not all were explained. (Brca1 results in DNA damage). Slide#98: A patient may complain of a metastasis without knowing where is the primary cancer, this microarray classifies types of cancers looking at expression profile so we can tell which is the primary tumor. Slide#99: Triple-negative breast cancer, i.e: 1) ER -ve (estrogen receptor) 2)PR -ve (progesterone receptor) 3)HER2 -ve This type of cancer is too aggressive chemotherapy. A new trend of understanding the triple ve breast cancer to give better treatment, by analyzing expression profile found out that there are 6 different classes of it based on distinct gene set expression! e.g. metabolism, androgen receptor, stem cell genes related triple negative breast cancer. Solution: give 2 or 3 types of cancer therapy. e.g. of personalized medicine: a lady with breast cancer. Suspect: 1) Estrogen receptor mutation, overexpression, amplification, hyperactivity give ER (estrogen receptor) inhibitor.
Slide#100: Hallmark of cancers: Only the bold line points were explained with example of each. Notes: Cancer cells are recognized and killed by the immune system (because they carry different protein and mutations). Some cells can avoid immune destruction by suppression and reduction of immune system, e.g. fake proteins that can inactivate immune cells. العلم نور وتشريف لصاحبه فاطلب هديت فنون العلم واألدبا العلمكنز وذخر ال فناء له نعم القرين إذا ما صاحب صحبا