PMID- 28860039
OWN - NLM
STAT- MEDLINE
DCOM- 20180712
LR  - 20180720
IS  - 1879-0542 (Electronic)
IS  - 0165-2478 (Linking)
VI  - 190
DP  - 2017 Oct
TI  - The use of BirA-BAP system to study the effect of US2 and US11 on MHC class I 
      heavy chain in cells.
PG  - 233-239
LID - S0165-2478(17)30281-X [pii]
LID - 10.1016/j.imlet.2017.08.027 [doi]
AB  - Biotinylation has been extensively used for antibody tagging, affinity-based 
      purification, and in protein/DNA-protein interaction studies. Here we describe 
      the use of biotinylation to study the turn-over of proteins in cells. We use the 
      prokaryotic biotin ligase (BirA) to biotinylate the human leukocyte antigen 
      (HLA)-A2 (A2) heavy chain (HC), which was engineered to contain a biotin acceptor 
      peptide (BAP). Controlled availability of biotin in combination with 
      visualization using streptavidin-conjugated peroxidase made it possible to detect 
      biotinylated BAP-A2. Further, we exploited the effects of human cytomegalovirus 
      (HCMV) unique short (US) proteins US2 and US11 on the turn-over of BAP-A2 HC. The 
      full-length BAP-A2 HC and its mutants lacking either the cytosolic tail 
      (tail-less) or both the transmembrane and cytosolic regions (soluble) were 
      expressed via recombinant adenoviruses (rAd). The effect of US2, US11 and a 
      control HCMV protein US9, also expressed via rAd, on each of the BAP- A2 forms 
      was assessed. Experiments using this system showed that US2 and US11 cause 
      proteasome-mediated degradation of full-length BAP-A2 HC but only US2 could cause 
      degradation of tail-less BAP-A2. The results demonstrate that the technique of 
      biotinylation can be used to study protein turn-over in cells.
CI  - Copyright (c) 2017 European Federation of Immunological Societies. Published by 
      Elsevier B.V. All rights reserved.
FAU - Gauthami, S
AU  - Gauthami S
AD  - Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad, 500078, 
      India.
FAU - Kumar, Deepak
AU  - Kumar D
AD  - Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad, 500078, 
      India.
FAU - SivaSai, K S R
AU  - SivaSai KSR
AD  - Sreenidhi Institute of Science & Technology, Yamnampet, Ghatkesar, Hyderabad, 
      501301, India.
FAU - Hegde, Nagendra R
AU  - Hegde NR
AD  - Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad, 500078, 
      India. Electronic address: hegde@niab.org.in.
LA  - eng
PT  - Journal Article
DEP - 20170830
PL  - Netherlands
TA  - Immunol Lett
JT  - Immunology letters
JID - 7910006
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (HLA-A2 Antigen)
RN  - 0 (Heat-Shock Proteins)
RN  - 0 (RNA-Binding Proteins)
RN  - 0 (Repressor Proteins)
RN  - 0 (US11 protein, herpesvirus)
RN  - 0 (US2 protein, Varicellovirus)
RN  - 0 (Viral Envelope Proteins)
RN  - 0 (Viral Proteins)
RN  - EC 3.4.21.92 (Endopeptidase Clp)
RN  - EC 3.6.1.3 (ClpB protein, E coli)
RN  - EC 6.3.- (Carbon-Nitrogen Ligases)
RN  - EC 6.3.4.15 (birA protein, E coli)
SB  - IM
MH  - Adenoviridae/genetics
MH  - Carbon-Nitrogen Ligases/*genetics
MH  - Cytomegalovirus/*physiology
MH  - Endopeptidase Clp/*genetics
MH  - Escherichia coli/*genetics
MH  - Escherichia coli Proteins/*genetics
MH  - Genetic Engineering
MH  - Genetic Vectors/genetics
MH  - HEK293 Cells
MH  - HLA-A2 Antigen/*genetics/metabolism
MH  - Heat-Shock Proteins/*genetics
MH  - Humans
MH  - Microorganisms, Genetically-Modified
MH  - Mutation/genetics
MH  - Proteolysis
MH  - RNA-Binding Proteins
MH  - Repressor Proteins/*genetics
MH  - Viral Envelope Proteins
MH  - Viral Proteins/*metabolism
OTO - NOTNLM
OT  - BAP tagging
OT  - Biotinylation
OT  - HCMV
OT  - MHC
OT  - Protein turn-over
EDAT- 2017/09/02 06:00
MHDA- 2018/07/13 06:00
CRDT- 2017/09/02 06:00
PHST- 2017/06/15 00:00 [received]
PHST- 2017/08/06 00:00 [revised]
PHST- 2017/08/25 00:00 [accepted]
PHST- 2017/09/02 06:00 [pubmed]
PHST- 2018/07/13 06:00 [medline]
PHST- 2017/09/02 06:00 [entrez]
AID - S0165-2478(17)30281-X [pii]
AID - 10.1016/j.imlet.2017.08.027 [doi]
PST - ppublish
SO  - Immunol Lett. 2017 Oct;190:233-239. doi: 10.1016/j.imlet.2017.08.027. Epub 2017 
      Aug 30.