Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase.
Anal Chem
; 96(21): 8721-8729, 2024 05 28.
Article
in En
| MEDLINE
| ID: mdl-38683735
ABSTRACT
Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein-Tyrosine Kinases
/
Escherichia coli Proteins
/
Protein Array Analysis
/
Escherichia coli
Language:
En
Journal:
Anal Chem
Year:
2024
Document type:
Article
Affiliation country:
Taiwán
Country of publication:
Estados Unidos