Short note for Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization

Note for myself on: Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization
(doi: 10.1042/BJ20131067)

1. Investigate the function of RNF167/RNF13 at the cellular level
 -- PA domain in these two proteins were shown to function as endosomal recycling processes
-- using the information from a functional study in fruitfly (homolog) and apply to the human cell
2. Using the information from COSMID to identify the mutations that could contribute to the cancer
3. Identified those mutations in human cell line// there are two separate sets;
-- PA domain function --> endosomal localization
-- RING domain function --> E3 ubiquitin ligase
It works independently but coordinatingly to get the correct function within the cell --> right place and right time to function.

Cell line;
Using HEK-293 to study the function

Antibodies;
FK2 (recognize K29-, K48-, K63-linked poly ubiquitinylated and mono ubiquitinylated proteins but not free ubiquitin)-- marker for ubiquitination process
EEA1 (Early Endosome Antigen 1) -- marker for endosome vesicle
GRP78/BiP -- marker for major endoplasmic reticulum (ER) chaperone protein

Fluorescence protein;
RNF13-GFP
RNF167-GFP
Rab5-DsRed (endosome marker)
Rab11-DsRed (endosome marker)



Information retrieved from COSMID and combining with the protein domains


Amino acid alignment of PA(green)-TM(orange)-RING(purple)


B --> PA domain and showing the conserved amino acids that may play role in tumor RNF167/13(red), in dimer interface (arrowhead area - blue AA)
C-->crystal structure of GRAIL PA domain; PDB-3ICU


The combination of functional studies at the same time by using IF, to indicate the function of PA as endocytic regulation and RING as ubiquitination.

AAs are responsible for localization of RNF167/RNF13 -- P (PA), R (RING)