Lab 1 - Laboratory Practices and Data Mining

Question 1

Explain the biological function of the enzyme PTEN in mammalian systems

Answer 1

The PTEN gene provides instructions for making an enzyme that is found in almost all tissues in the body. The enzyme acts as a tumor suppressor, which means that it helps regulate cell division by keeping cells from growing and dividing (proliferating) too rapidly or in an uncontrolled way.

Question 2

How did cutting edge technology like biotechnology fundamentally change societies?

Answer 2

Through the capacity to provide important benefits of health, the environment, and economics

Question 3

In particular, what is this lab simulating?

Answer 3

The initial characterization of a tumour suppressor enzyme using various biotechnology approaches

Question 4

What tumour suppressor enzyme will we be studying?

Answer 4

Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN)

Question 5

When was the PTEN gene isolated?

Answer 5

During a screen for deleted genes in cancers that were located on the frequently mutated chromosome 10

Question 6

What advanced cancers has PTEN been shown to be mutated in?

Answer 6

Breast, brain, prostate, and kidney

Question 7

What will be the focus of this laboratory?

Answer 7

The protein sequence analysis of the gene

Question 8

What will be the focus of the laboratories for the remainder of the semester?

Answer 8

The initial characterization of the PTEN protein

Question 9

In this lab, the bioinformatics databases contained on what will be used?

Answer 9

The National Centre for Biotechnology Information’s servers (NCBI)

Question 10

When was NCBI established? By who? For what purpose?

Answer 10

In 1988 by the US government as a national resource for molecular biology information

Question 11

What does the NCBI currently do?

Answer 11

• Creates public databases
• Conducts research in computational biology
• Develops software tools for analyzing genome data
• Disseminates biomedical information with the goal of increasing the level of understanding associated with molecular processes that impact human health and disease

Question 12

What is an effective method for analyzing protein sequences?

Answer 12

Examine conserved domains between a protein of interest and group of protein homologs

Question 13

What is a conserved domain (CD)?

Answer 13

A region within a protein sequence that demonstrates a statistically significant correlation with a region on a different protein

Question 14

What about CDs can be determined by comparative analysis?

Answer 14

Recurring units in polypeptide chains (sequence and structure motifs)

Question 15

How can molecular evolution use CDs?

Answer 15

As building blocks which may be recombined in different arrangements to make different proteins with different functions

Question 16

By using CDs, what can we learn about PTEN?

Answer 16

• The function of the enzyme
• Locate functional domains
• Establish evolutionary relationships across protein families
• Interpret mutation studies
• Make predictions on the structure of a protein of unknown structure

Question 17

What does the conserved domain database (CDD) contain?

Answer 17

Sequence alignments that define the features that are conserved within each domain family

Question 18

What does the CDD serve as?

Answer 18

A classification resource that groups proteins based on the presence of these predefined domains

Question 19

What do CDD entries often give?

Answer 19

The name of the domain family and a description of the role of conserved residues in binding or catalysis

Question 20

What are CDs displayed in and what do they link to?

Answer 20

MMDB structure summaries and link to a sequence alignment showing other proteins in which the domain is conserved, which may provide clues on protein function

Question 21

How is CD-Search designed to interact with the NCBI databases?

Answer 21

To perform multiple sequence alignments and detailed statistical analysis using the BLAST software suite - an NCBI search algorithm

Question 22

How does CD-Search use BLAST statistics?

Answer 22

Evaluates the statistical significance of query - subject alignments and returns E-values (expectation values), which can be used in your analysis

Question 23

What does the CD-Search using BLAST statistics database program report?

Answer 23

A graphical summary of each search, a list of hits (matches), and a pair wise alignment between your query and the conserved domains of other proteins

Question 24

What is Ensembl?

Answer 24

A joint project between EMBL-EBI and the Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes

Question 25

What is the protocol for “Bioinformatic survey of PTEN”?

Answer 25

1) Go to NCBI
2) Select “protein”
3) Search across all the NCBI databases for “phosphatase and tensin homolog”
4) Select “Homo sapiens”
5) Select “gene” and “find items”
6) Search for the name/Gene ID “PTEN” and click the title
7) Return to results page and check off the ID box for PTEN
8) Select “database” then “conserved domain” then “options” then “conserved domain (concise), then “find items”
9) Explore predicted conserved domains associated with PTEN

Question 26

What is the protocol for “Ensembl”?

Answer 26

1) Go to the Ensembl database
2) Select “human” database and search for “PTEN”
3) Restrict the category to “gene”
4) Select the entry PTEN (human gene) and explore the various options to learn about the experimental knowledge on PTEN
5) Investigate the protein’s description of physiological function, transcript structure, and orthologues

Question 27

Results: What is the official symbol, name, additional designations, and chromosome location of PTEN?

Answer 27

• PTEN
• Phosphatase and tensin homolog
• B2S, DEC, CWS1, GLM2, MHAM, TEP1, MMAC1, PTEN1, 10q23dek, PTENbeta
• 10q23.31

Question 28

Results: Based on your gene and conserved domain search, what did the program predict the biological function of PTEN to be?

Answer 28

1) Conserved protein domain family: PTP_DSP_cys. Made up of cys-based phosphatases and distinguished by a CxxxxxR conserved catalytic loop. Protein tyrosine phosphatases aid in responding to cells where there are physiological and pathological changes to their environment.
2) Conserved protein domain family: PTEN_C2. The C2 domain of PTEN is a phosphatidyl-inositol triphosphate phosphatase which has a CBR3 loop critical for membrane binding by strategically putting the catalytic part of the protein onto the membrane.
- Overall, it’s biological function is to preferentially dephosphorylate phosphoinositide substrates and negatively regulate Akt

Question 29

Results: What conserved domains produced the best alignment of PTEN? What signature motif sequence provided the strongest evidence for the prediction?

Answer 29

• PTP_DSP_cys and PTPc because they are the highest on the list of the curated CD hierarchy, meaning they have the most similarity in terms of sequence alignment
• xxCxxxRx is the signature motif sequence that provides the strongest evidence for this prediction

Question 30

Results: What is an orthologue?

Answer 30

Genes that have evolved when populations of species have separated. The genes are therefore related by a common ancestor.

Question 31

Results: What are some of PTENs orthologues?

Answer 31

Rodents, lagomorphs, tree shrews, ungulates, and ray-finned fish

Question 32

Results: How many residues does PTEN have and what is its molecular weight?

Answer 32

18 residues for PTEN. The molecular weight for TEN-201 is 47,166.29 g.mol or 47.16629 kDa

Question 33

Results: In the human genome, what chromosome does the PTEN gene reside? Where specifically on this chromosome is the gene located?

Answer 33

Chromosome 10, specifically, located from 87863625 base pairs to 87971930 base pairs on the forward strand

Question 34

Results: What is the description of PTEN based on the “protein family ID” in Ensembl?

Answer 34

PTEN contains a tensin like domain and catalytic domain and it functions as a tumour suppressor by negatively regulating the Akt/PKB signalling pathway by dephosphorylating phosphoinositide substrates and negatively regulating intracellular levels of phosphatidylinositol-3,4,5-triphosphate

Question 35

Results: From Ensemble, what are the two domain superfamilies and their SCOP (Structural Classification Of Proteins) classification and functional annotation?

Answer 35

1) C2 domain
–> Root: SCOP hierarchy in superfamily
–> Class: All beta proteins
–> Fold: C2 domain like
2) Phosphotyrosine protein phosphatases II
–> Root: SCOP hierarchy in superfamily
–> Class: Alpha and beta proteins
–> Fold: (Phosphotyrosine protein) phosphatases II

Question 36

Results: Provide an explanation of what the function of the C2 domain is pertaining to the functional activity of PTEN.

Answer 36

Helps to position the catalytic part of the protein onto the membrane which negatively regulates the signal transduction and contributes to PTENs tumour suppressing capabilities

Question 37

Results: List the reference that was the primary study that determined the physiological substrate for PTEN.

Answer 37

Hopkin, K. (1998). A surprising function for the PTEN tumor suppressor. Science. 282(5391), 1027-1030.