Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays

Huang, Che-Fan; Mrksich, Milan

doi:10.1021/acscombsci.9b00152

Cited by 12 publications

(12 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Remarkably, the rule we describe—where PTPs disfavor basic residues proximal to pY—is surprisingly general. In this work, we show how this insight can explain signaling in β-cat mutants and in previous work we addressed its role in insulin receptor 22 . We note that our database search identified over 6000 relevant cancer mutations that could employ this missense mutation/PTP restriction mechanism, and suggests that this mechanism can have a broad relevance in understanding mutations that underlie cancers.…”

Section: Discussionmentioning

confidence: 84%

“…In recent work, we used peptide arrays and SAMDI-MS 15 – 21 (self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry) to profile the specificities of 22 phosphatases including PTP1B, TC-PTP, MEG1, SHP1, PTPN7, MEG2, SHP2, PTPN12, PTPN13, PTPN14, PTPRA, PTPbeta, CD45, PTPRE, RPTPG, DEP1, PTPmu, PTPsigma, PRL1-3 and hALP (Fig. 1 ) 22 . We found that most of the PTPs lack activity towards substrates that have an arginine (R) or lysine (K) residue adjacent to the phosphotyrosine (pY).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates

Huang

Gottardi

Mrksich

2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell–cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.

show abstract

Section: Discussionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates

Huang

Gottardi

Mrksich

2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…After purification, the RST can be removed using either these proteases or SUMO protease (for RST N ). Treatment with TEV leaves a GC scar at the N-terminus, where the cysteine is included for SAMDI (self-assembled monolayers on gold for matrix-assisted laser desorption/ionization) mass spectroscopy [ 47 , 48 ].…”

Section: Resultsmentioning

confidence: 99%

Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions

et al. 2022

View full text Add to dashboard Cite

RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a class of pharmaceutically-relevant natural products expressed as precursor peptides before being enzymatically processed into their final functional forms. Bioinformatic methods have illuminated hundreds of thousands of RiPP enzymes in sequence databases and the number of characterized chemical modifications is growing rapidly; however, it remains difficult to functionally express them in a heterologous host. One challenge is peptide stability, which we addressed by designing a RiPP stabilization tag (RST) based on a small ubiquitin-like modifier (SUMO) domain that can be fused to the N- or C-terminus of the precursor peptide and proteolytically removed after modification. This is demonstrated to stabilize expression of eight RiPPs representative of diverse phyla. Further, using Escherichia coli for heterologous expression, we identify a common set of media and growth conditions where 24 modifying enzymes, representative of diverse chemistries, are functional. The high success rate and broad applicability of this system facilitates: (i) RiPP discovery through high-throughput “mining” and (ii) artificial combination of enzymes from different pathways to create a desired peptide.

show abstract

“…Solid-phase peptide synthesis (SPPS) was performed as described previously . Briefly, SPPS was performed on Rink-amide resin (10 mg) housed in 96-well filter plates.…”

Section: Methodsmentioning

confidence: 99%

Efficient Enzymatic Incorporation of Dehydroalanine Based on SAMDI-Assisted Identification of Optimized Tags for OspF/SpvC

et al. 2022

Self Cite

View full text Add to dashboard Cite

Site-specific modification of proteins has important applications in biological research and drug development. Reactive tags such as azide, alkyne, and tetrazine have been used extensively to achieve the abovementioned goal. However, bulky side-chain “ligation scars” are often left after the labeling and may hinder the biological application of such engineered protein products. Conjugation chemistry via dehydroalanine (Dha) may provide an opportunity for “traceless” ligation because the activated alkene moiety on Dha can then serve as an electrophile to react with radicalophile, thiol/amine nucleophile, and reactive phosphine probe to introduce a minimal linker in the protein post-translational modifications. In this report, we present a mild and highly efficient enzymatic approach to incorporate Dha with phosphothreonine/serine lyases, OspF and SpvC. These lyases originally catalyze an irreversible elimination reaction that converts a doubly phosphorylated substrate with phosphothreonine (pT) or phosphoserine (pS) to dehydrobutyrine (Dhb) or Dha. To generate a simple monophosphorylated tag for these lyases, we conducted a systematic approach to profile the substrate specificity of OspF and SpvC using peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. The optimized tag, [F/Y/W]-pT/pS-[F/Y/W] (where [F/Y/W] indicates an aromatic residue), results in a ∼10-fold enhancement of the overall peptide labeling efficiency via Dha chemistry and enables the first demonstration of protein labeling as well as live cell labeling with a minimal ligation linker via enzyme-mediated incorporation of Dha.

show abstract

Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays

Cited by 12 publications

References 62 publications

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates

Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions

Efficient Enzymatic Incorporation of Dehydroalanine Based on SAMDI-Assisted Identification of Optimized Tags for OspF/SpvC

Contact Info

Product

Resources

About