Extendable stapling of unprotected peptides by crosslinking two amines with o-phthalaldehyde

Peptide modification methods that do not rely on the cysteine residue are underdeveloped, and their development could greatly expand the current toolbox for peptide chemistry. During the course of preliminary investigations into the classical ortho-phthalaldehyde (OPA)-amine-thiol condensation reaction, we found that in the absence of thiol, OPA readily condenses with two primary alkyl amines to form a class of underexplored isoindolin-1-imine compounds under mild aqueous conditions. From the intramolecular version of this OPA-2amines reaction, an efficient and selective methodology using mild reaction conditions has been developed for stapling unprotected peptides via crosslinking of two amino groups in both an end-to-side and side-to-side fashion. The stapling method is superfast and broadly applicable for various peptide substrates with the reacting amino groups separated by a wide range of different amino acid units. The macrocyclization reactions of selected substrates are completed within 10 seconds at 5 mM concentration and within 2 minutes at 50 μM concentration. Importantly, the resulting cyclized peptides with an isoindolinimine linkage can be extended in a one-pot sequential addition manner with several different electron-deficient π electrophiles, thereby generating more complex structures.

Functional interplay between arginyl-tRNA synthetases and arginyltransferase

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance between ATE1, RARS and translation are unknown. Here we addressed the functional interplay between these mechanisms using intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We find that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1 redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence between translation, arginyl-tRNA synthesis, and arginylation.

Prolylcarboxypeptidase Promotes IGF1R/HER3 Signaling and Is a Potential Target to Improve Endocrine Therapy Response in Estrogen Receptor Positive Breast Cancer

Background Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked PRCP to blood-pressure and appetite control through its ability to cleave peptide substrates such as angiotensin II and a-MSH. A potential role for PRCP in cancer has to date not been widely appreciated. Endocrine therapy resistance in breast cancer is an enduring clinical problem mediated in part by aberrant receptor tyrosine kinase (RTK) signaling. We previously found PRCP overexpression promoted tamoxifen (TAM) resistance in estrogen receptor-positive (ER+) breast cancer cells. Currently we tested the potential association between PRCP with breast cancer patient outcome and RTK signaling, and tumor responsiveness to endocrine therapy. Methods We analyzed PRCP protein expression by IHC staining of ER+ breast cancer samples and PRCP gene expression in clinical databases and used Kaplan-Meier survival curves to determine the significance of PRCP expression correlation with recurrence free survival and overall survival. We analyzed PRCP-regulated IGF1R/HER3 signaling using immunoblotting in the ER+ MCF7 cell line. We analyzed the therapeutic effect of a PRCP specific inhibitor (PRCPi) and/or endoxifen on tumor growth of ER+ PDX tumors and MCF7 tumors in immunocompromised mice. ResultsWe found high PRCP protein levels in tumors associates with worse outcome and earlier recurrence in breast cancer patients, including patients treated with TAM. Analyses of clinical databases showed that PRCP expression correlates with IGF1 and NRG1 expression and their target genes and earlier recurrence in endocrine-treated ER+/HER2- breast cancer patients. Overexpression of PRCP increased IGF1R/HER3 signaling. PRCPi blocked IGF1R/HER3 signaling and enhanced the response of ER+ breast cancer tumors in mice to endoxifen, the active metabolite of TAM. ConclusionsPRCP is an adverse prognostic marker in breast cancer and a potential target to improve endocrine therapy in ER+ breast cancers.

Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

Proteasome Activator 28γ (PA28γ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28γ functions in protein degradation remains unclear. Though PA28γs mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28γ regulates proteasome function. Here, we rigorously and definitively show that PA28γ uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28γ-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28γ also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28γ-20S proteasome complex at 4.4A validating it's expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28γ functions by allosterically upregulating the T-L like site in the 20S proteasome.

Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

Traceless enzymatic protein synthesis without ligation sites constraint

Protein synthesis and semisynthesis offer immense promise for life science and have impacted pharmaceutical innovation. Nevertheless, the absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets. Here we introduce the protein activation and ligation with multiple enzymes (PALME) platform designed for the sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with the preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization, and protein N/C-terminal or internal functionalization. This methodology's practical utility was demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. Together, the modular PALME platform exhibits unprecedented broad accessibility for the traceless protein synthesis and functionalization and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

Facile access to C-glycosyl amino acids and peptides via Ni-catalyzed reductive hydroglycosylation of alkynes

C-Glycosyl peptides/proteins are metabolically stable mimics of the native glycopeptides/proteins bearing O/N-glycosidic linkages, and are thus of great therapeutical potential. Herein, we disclose a protocol for the syntheses of vinyl C-glycosyl amino acids and peptides, employing a nickel-catalyzed reductive hydroglycosylation reaction of alkyne derivatives of amino acids and peptides with common glycosyl bromides. It accommodates a wide scope of the coupling partners, including complex oligosaccharide and peptide substrates. The resultant vinyl C-glycosyl amino acids and peptides, which bear common O/N-protecting groups, are amenable to further transformations, including elongation of the peptide and saccharide chains.

Evaluation of the effects of phosphorylation of synthetic peptide substrates on their cleavage by caspase-3 and -7

Caspases are a family of enzymes that play roles in cell death and inflammation. It has been suggested that in the execution phase of the apoptotic pathway, caspase-3, -6 and -7 are involved. The substrate specificities of two proteases (caspases 3 and 7) are highly similar, which complicates the design of compounds that selectively interact with a single enzyme exclusively. The recognition of residues other than Asp in the P1 position of the substrate by caspase-3/-7 has been reported, promoting interest in the effects of phosphorylation of amino acids in the direct vicinity of the scissile bond. To evaluate conflicting reports on this subject, we synthesized a series of known caspase-3 and -7 substrates and phosphorylated analogs, performed enzyme kinetic assays and mapped the peptide cleavage sites using internally quenched fluorescence peptide substrates. Caspases 3 and 7 will tolerate pSer at the P1 position but only poorly at the P2’ position. Our investigation demonstrates the importance of peptide length and composition in interpreting sequence/activity relationships. Based on the results, we conclude that the relationship between caspase-3/-7 and their substrates containing phosphorylated amino acids might depend on the steric conditions and not be directly connected with ionic interactions. Thus, the precise effect of phospho-amino acid residues located in the vicinity of the cleaved bond on the regulation of the substrate specificity of caspases remains difficult to predict. Our observations allow to predict that natural phosphorylated proteins may be cleaved by caspases, but only when extended substrate binding site interactions are satisfied.