Abstract
Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1920-1936 |
| Number of pages | 17 |
| Journal | Journal of Proteome Research |
| Volume | 14 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 3 2015 |
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Keywords
- AMPylation
- interactome
- LidA
- nucleic acid programmable protein array (NAPPA)
- Rab1
- SidM
ASJC Scopus subject areas
- Biochemistry
- Chemistry(all)
Cite this
Host-pathogen interaction profiling using self-assembling human protein arrays. / Yu, Xiaobo; Decker, Kimberly B.; Barker, Kristi; Neunuebel, M. Ramona; Saul, Justin; Graves, Morgan; Westcott, Nathan; Hang, Howard; LaBaer, Joshua; Qiu, Ji; Machner, Matthias P.
In: Journal of Proteome Research, Vol. 14, No. 4, 03.04.2015, p. 1920-1936.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Host-pathogen interaction profiling using self-assembling human protein arrays
AU - Yu, Xiaobo
AU - Decker, Kimberly B.
AU - Barker, Kristi
AU - Neunuebel, M. Ramona
AU - Saul, Justin
AU - Graves, Morgan
AU - Westcott, Nathan
AU - Hang, Howard
AU - LaBaer, Joshua
AU - Qiu, Ji
AU - Machner, Matthias P.
PY - 2015/4/3
Y1 - 2015/4/3
N2 - Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.
AB - Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.
KW - AMPylation
KW - interactome
KW - LidA
KW - nucleic acid programmable protein array (NAPPA)
KW - Rab1
KW - SidM
UR - http://www.scopus.com/inward/record.url?scp=84926512827&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84926512827&partnerID=8YFLogxK
U2 - 10.1021/pr5013015
DO - 10.1021/pr5013015
M3 - Article
C2 - 25739981
AN - SCOPUS:84926512827
VL - 14
SP - 1920
EP - 1936
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 4
ER -