| 1. |
- Gould, Cathryn M, et al.
(författare)
-
ELM : the status of the 2010 eukaryotic linear motif resource.
- 2010
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38
-
Tidskriftsartikel (refereegranskat)abstract
- Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.
|
|
| 2. |
- Karlsson, O. Andreas, et al.
(författare)
-
Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (K-d = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.
|
|
| 3. |
- Kassa, Eszter
(författare)
-
Exploration of analytical methods to study motif-mediated host-virus protein-protein interactions
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are responsible for countless processes in living creatures, but most often they do not perform these tasks alone. Rather, they engage in interactions with other proteins, creating whole protein-protein interaction (PPI) networks. Some of these interactions are formed between a folded protein domain and a short linear motif (SLiM), which is a small, 3-10 amino acid long stretch usually in the intrinsically disordered regions of proteins. These interactions tend to be low-to-medium affinity and transient, therefore their capture requires special tools. Furthermore, viruses often hijack the human cellular machinery through PPIs as they have limited genomes and are obligate cellular parasites. Therefore, the investigation of viral-host PPIs is of great importance and can lead to the development of novel antivirals.In my thesis, I used mostly peptide-based and mass spectrometry (MS) techniques to validate and further explore motif-based PPIs. The main objectives were to: i) evaluate and compare synthetic peptide-based pulldown approaches, ii) validate and further explore the interaction between viral peptides and human polyadenylate-binding protein (PABP) using green fluorescent protein (GFP)-tagged peptide repeats, iii) confirm interactions, define and refine human interaction motifs that engage in interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins by employing peptide SPOT (synthetic peptide arrays on membrane support technique) arrays and alanine scanning, iv) investigate the change in the interactome of the nuclear pore complex protein 153 (NUP153) between uninfected and tick-borne encephalitis virus (TBEV)-infected states using GFP-tagged full-length protein for pulldown.First, we explored the potential of affinity purification-mass spectrometry (AP-MS) and protein interaction screen on peptide matrix (PRISMA) to capture SLiM-based PPIs. The peptide pulldown approach proved to be more applicable over a wide range of affinities and interactions, however, protein concentration and the local concentration of presented motifs were limiting factors in certain cases. We then investigated SLiM-based interactions between RNA-viruses and human proteins. Here, using green fluorescent-peptide pulldowns I confirmed the interaction between viral peptides and the human poly-A binding protein. Next, we uncovered that some human SLiMs interact with SARS-CoV-2 proteins, and I was able to highlight the interaction motif using peptide arrays when only a handful of peptides were available. Lastly, I identified different enriched proteins in NUP153-pulldowns from mock-infected and TBEV-infected cell lysate, that were complementary to the changes observed with other techniques.In conclusion, I explored a range of techniques that are valuable in the validation of PPIs, which is crucial in combination with high-throughput approaches. As more and more SLiM-based interactions are explored and predicted, the value of these tools continues to increase.
|
|
