Detection of Postranslational Modifications

NAPPA can be coupled with a variety of biochemical modification systems to identify proteins that add post-translational modifications to other proteins or to identify protein targets. Furthermore, once developed, these systems provide an ideal method for screening drugs or other small molecule inhibitors. We have three main projects studying different post-translational modifications in the center:

  1. Phosphorylation and its applications in Chronic Myelogenous Leukemia
  2. AMPylation and its role in bacterial pathogenesis
  3. Citrullination and its role in the pathogenesis of rheumatoid arthritis


Using protein microarrays to examine small molecule inhibitory specificity: Phosphorylation

Investigator: Fernanda Festa, Ph.D.

Collaborators: Nathaniel Gray Ph.D. and Jianming Zhang, Ph.D. (Dana Farbar Cancer Institute)

Disease Application: Chronic Myelogenous Leukemia

Protein kinases are important players in all aspects of cell biology, regulating cell signaling pathways that ultimately define the cell fate. The understanding of this key class of proteins has been proven to be fundamental for the treatment and diagnosis of many diseases, especially cancer. Nonetheless, many kinases still have unknown substrates and/or modulators, and a more comprehensive understanding of this class of proteins has the potential to improve disease outcome. The protein array platform is very flexible and many distinct applications can be envisioned, including the screening of new substrates, drug sensitivity and protein/protein interaction, among others.

We initially tested the ability to detect protein phosphorylation on the NAPPA array by creating a cloned NAPPA-compatible plasmid collection of all human kinases. Using kinase NAPPA arrays we demonstrated that the kinases on the array display autophosphorylation activity. To demonstrate this, anti-pTyr antibodies detect phosphotyrosines on the kinases on the arrays, which disappear after treatment with phosphatase and then returns after incubation in kinase buffer and ATP (see image). Ongoing experiments are using kinase NAPPA arrays to study the effect of specific kinase inhibitors (small molecules) on the kinase activity and determine the small molecule selectivity and efficacy among many tested kinases, all performed in a single experiment.



Investigator: Xiaobo Xu, Ph.D.

Collaborators: Andrew Woolery and Kim Orth, Ph.D. (UT Southwestern Medical Center), Howard Hang, Ph.D. (The Rockefeller University), Kimberly Decker, Ph.D. and Matthias P. Machner, Ph.D. (NICHD), Michael L. Syring and Konstantinos Petritis, Ph.D. (TGen) and Shelley Haydel, Ph.D.(CIDV, Biodesign)

Disease Application: Bacterial Infection

In recent years, a new type of post-translational modification (PTM), AMPylation, has emerged as a fundamental mechanism regulating protein-protein interactions and cell signaling during bacterial pathogenesis. The AMPylation reaction is mediated by virulence factors from bacteria that are secreted into the host cells and can transfer AMP from ATP to the Tyrosine or Threonine group of GTPases. The result of AMPylation is disruption of GTPase binding to downstream effectors such as PAK1 resulting in cell toxicity. AMPylation domains are conserved in both prokaryotic and eukaryotic organisms; therefore, we expect that protein AMPylation plays an important role in a wide range of cellular processes. However, our understanding of AMPylation is limited and identifying new protein AMPylators and their substrates will help illuminate the functional consequences of AMPylation.

To address this question, we developed a non-radioactive assay for the detection of AMPylated proteins using NAPPA. With this method we screened 10,000 unique human proteins with two known bacterial AMPylators, VopS and IbpAFic2, and identified new GTPase and non-GTPase substrates dramatically expanding our knowledge of the substrates of these enzymes (see image). These results suggest that this approach could be extended to identify novel substrates of other AMPylators in different species or with different domains.


Investigator: Ji Qiu, Ph.D.

Collaborators: Jerry Nepom, Ph.D. (Benaroya Research Institute), and Jane Buckner (Benaroya Research Institute)

Disease Application: Rheumatoid Arthritis

We are also interested in studying antibodies against citrullinated proteins in rheumatoid arthritis in collaboration with Drs. Jerry Nepom and Jane Buckner at the Benaroya Research Institute.  Anti-citrulline antibodies are specific and predictive of rheumatoid arthritis (RA).  Seropositivity against citrulline is clinically assayed using cyclic citrullinated peptide (CCP) ELISA.  Despite being an excellent proxy assay for diagnosis, anti-CCP positivity does not reveal any information about the actual underlying antigens that elicited the immune response.  Recent studies demonstrated the value of identifying autoantibodies to particular antigens in the elucidation of RA etiology.  Furthermore, the use of specific citrullinated antigens could improve diagnostic performance.  Unfortunately, only a few citrullinated antigens have been discovered in the past several decades.  Traditional protein immuno-chemistry methods to identify citrullinated antigens suffer drawbacks such as low throughput, poor reproducibility, inadequate quantification and low resolution.  Commercial protein arrays are expensive, lack equal representation of candidate antigens and are not compatible with post-translational modifications such as citrullination, which requires harsh conditions.  Our overarching goal is to discover additional antigens, when citrullinated, can be recognized by antibodies in RA patients at the proteome level.  Identification of these citrullinated antigens will not only help understand the disease pathogenesis but also improve diagnosis and patient stratification.