Contact
Quote

Home » Publications

Publications

Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

Filter by
Filtered (9)
Filters
Show (9)
Cancel
Showing most recent

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

Pras, Anita; Houben, Bert; Aprile, Francesco A.; Seinstra, Renée; Gallardo, Rodrigo; Janssen, Leen; Hogewerf, Wytse; Gallrein, Christian; De Vleeschouwer, Matthias; Mata-Cabana, Alejandro; Koopman, Mandy; Stroo, Esther; de Vries, Minke; Louise Edwards, Samantha; Kirstein, Janine; Vendruscolo, Michele; Falsone, Salvatore Fabio; Rousseau, Frederic; Schymkowitz, Joost; Nollen, Ellen A. A.
EMBO J.
Nov 2021
10.15252/embj.2020107568
While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

Das, Aniruddha; Thapa, Pankaj; Santiago, Ulises; Shanmugam, Nilesh; Banasiak, Katarzyna; Dabrowska, Katarzyna; Nolte, Hendrik; Szulc, Natalia A.; Gathungu, Rose M.; Cysewski, Dominik; Krüger, Marcus; Dadlez, Michal; Nowotny, Marcin; Camacho, Carlos J.; Hoppe, Thorsten; Pokrzywa, Wojciech
Aug 2021
10.1101/2021.08.20.457118
The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

Zhang, Zhiqiang; Cottignie, Ines; Van Zeebroeck, Griet; Thevelein, Johan M.
Biochem J.
Jan 2021
10.1042/BCJ20200722
Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor–Sch9–TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor–Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

Probing peptide sequences on their ability to generate affinity sites in molecularly imprinted polymers

Piletska, Elena V; Guerreiro, Antonio; Mersiyanova, Margarita; Cowen, Todd; Canfarotta, Francesco; Piletsky, Stanislav S.; Karim, Kal; Piletsky, Sergey A.
Langmuir.
Dec 2019
10.1021/acs.langmuir.9b03410
An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake

Finnell, Jordan G.; Tsang, Tsz-Ming; Cryan, Lorna; Garrard, Samuel; Lee, Sai-Lun; Ackroyd, P. Christine; Rogers, Michael S.; Christensen, Kenneth A.
Jul 2019
10.1101/705459
Abstract Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. In addition to its role as an anthrax toxin receptor, CMG2 has been repeatedly shown to play a role in angiogenic processes. However, the molecular mechanism mediating observed CMG2-related angiogenic effects has not been fully elucidated. Previous studies have found that CMG2 binds type IV collagen (Col-IV), a key component of the vascular basement membrane, as well as other ECM proteins. Currently, no link has been made between these CMG2-ECM interactions and angiogenesis; however, ECM fragments are known to play a role in regulating angiogenesis. Here, we further characterize the CMG2-Col-IV interaction and explore the effect of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (non-collagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative binding epitope. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with sub-micromolar affinity (peptide S16, K d = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding, and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. We demonstrate that CMG2 specifically mediates endocytic uptake of S16, since CMG2-/- endothelial cells show markedly reduced S16 uptake, without reducing total endocytosis. Furthermore, we show that S16 reduces endothelial migration but not cell proliferation. Taken together, our data demonstrate that a Col IV-derived anti-angiogenic peptide acts via CMG2, suggesting a possible link between CMG2-Col IV interactions and angiogenesis.

Bacterial inhibitors

Xie, Hua
Jul 2019
Peptides related to certain portions of the arginine deiminase enzyme from the bacterium Streptococcus cristatus are provided that disrupt the formation and composition of biofilms containing the oral pathogen Porphyromonas gingivalis, and also modulate the virulence of P. gingivalis. Pharmaceutical compositions containing such peptides and method of using the same are disclosed.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
10.1038/s41598-017-17071-0
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

A recombinant BBSome core complex and how it interacts with ciliary cargo

Klink, Björn Udo; Zent, Eldar; Juneja, Puneet; Kuhlee, Anne; Raunser, Stefan; Wittinghofer, Alfred
Nov 2017
10.7554/eLife.27434
Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins involves the BBSome, an eight-membered protein complex that is recruited to ciliary membranes by the G-protein Arl6. BBSome malfunction leads to Bardet-Biedl syndrome, a ciliopathy with severe consequences. Short ciliary targeting sequences (CTS) have been identified that trigger the transport of ciliary proteins. However, mechanistic studies that relate ciliary targeting to BBSome binding are missing. Here we used heterologously expressed BBSome subcomplexes to analyze the complex architecture and to investigate the binding of GPCRs and other receptors to the BBSome. A stable heterohexameric complex was identified that binds to GPCRs with interactions that only partially overlap with previously described CTS, indicating a more complex recognition than anticipated. Arl6•GTP does not affect these interactions, suggesting no direct involvement in cargo loading/unloading.

Identification of Streptococcus cristatus peptides that repress expression of virulence genes in Porphyromonas gingivalis

Ho, Meng-Hsuan; Lamont, Richard J.; Xie, Hua
Sci Rep.
May 2017
10.1038/s41598-017-01551-4
Dental plaque is a complex multispecies biofilm, and is a direct precursor of periodontal disease. The virulence of periodontal pathogens, such as Porphyromonas gingivalis, is expressed in the context of this polymicrobial community. Previously, we reported an antagonistic relationship between Streptococcus cristatus and P. gingivalis, and identified arginine deiminase (ArcA) of S. cristatus as the signaling molecule to which P. gingivalis responds by repressing the expression and production of FimA protein. Here we demonstrate that direct interaction between P. gingivalis and S. cristatus is necessary for the cell-cell communication. Two surface proteins of P. gingivalis, PGN_0294 and PGN_0806, were found to interact with S. cristatus ArcA. Using a peptide array analysis, we identified several P. gingivalis-binding sites of ArcA, which led to the discovery of an 11-mer peptide with the native sequence of ArcA that repressed expression of fimbriae and of gingipains. These data indicate that a functional motif of ArcA is sufficient to selectively alter virulence gene expression in P. gingivalis, and PGN_0294 and PGN_0806 may serve as receptors for ArcA. Our findings provide a molecular basis for future rational design of agents that interfere with the initiation and formation of a P. gingivalis-induced pathogenic community.

Quote form