C-terminal Proline Research Articles

Prior disulfide bond-mediated Ser/Thr ligation.

In this work, we developed a novel strategy, prior disulfide bond-mediated Ser/Thr ligation (PD-STL), for the chemical synthesis of peptides and proteins. This approach combines disulfide bond-forming chemistry with Ser/Thr ligation (STL), converting intermolecular STL into intramolecular STL to effectively proceed regardless of concentrations. We demonstrated the effectiveness of PD-STL under high dilution conditions, even for the relatively inert C-terminal proline at the ligation site. Additionally, we applied this method to synthesize the N-terminal cytoplasmic domain (2-104) of caveolin-1 and its Tyr14 phosphorylated form.

Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.

Prolidase activity in women with polycystic ovarian syndrome undergoing assisted conception

Background: Prolidase is a manganese (Mn)-dependent cytosolic exopeptidase that degrades imidodipeptides with C-terminal proline or hydroxyproline. Prolidase recycling from imidodipeptides plays a critical role in collagen resynthesis and extracellular matrix (ECM) remodelling. Following an increase in gonadotropins, ovarian and follicular collagen undergo substantial degradation. Abnormal ovarian ECM composition is associated with polycystic ovary syndrome (PCOS). This study aimed to examine prolidase activity in the serum and follicular fluid (FF) of women undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) treatment, comparing those with PCOS to those with normal ovarian function.Methods: This prospective study enrolled 50 participants, of whom 44 were included. PCOS diagnosis followed the Rotterdam consensus criteria, with 20 patients constituting the study group. The control group comprised 24 individuals with mild-to-moderate male infertility. Prolidase enzyme activity in serum and FF was measured using the Chinard reagent via spectrophotometric analysis and compared between the groups.Results: Serum and FF prolidase levels were significantly lower in patients with PCOS (p < 0.05). A direct correlation was observed between serum and FF prolidase levels (p < 0.05). Although blastocyst quality scoring (BQS) significantly decreased in PCOS patients, no statistical difference was observed in the clinical pregnancy rate between the groups (p < 0.05) (p > 0.05). A negative correlation existed between serum prolidase levels and total antral follicle (AF) count (p < 0.05). Conversely, both serum and FF prolidase levels positively correlated with BQS (r = 0.574)(p < 0.05) (r = 0.650)(p < 0.05).Conclusions: Patients with PCOS showed lower serum and FF prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, potentially causing anovulation.

KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline- the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors- Krüppel‐like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor β1 (TGF-β1) signaling, since both KLF6 and Sp1 are key players in TGF-β1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-β1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-β1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-β1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Defining E3 ligase–substrate relationships through multiplex CRISPR screening

Specificity within the ubiquitin–proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates—and in particular the molecular features (degrons) that they recognize—remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin–proteasome system.

Prolidase Deficiency Causes Spontaneous T Cell Activation and Lupus-like Autoimmunity.

Prolidase deficiency (PD) is a multisystem disorder caused by mutations in the PEPD gene, which encodes a ubiquitously expressed metallopeptidase essential for the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. PD typically presents in childhood with developmental delay, skin ulcers, recurrent infections, and, in some patients, autoimmune features that can mimic systemic lupus erythematosus. The basis for the autoimmune association is uncertain, but might be due to self-antigen exposure with tissue damage, or indirectly driven by chronic infection and microbial burden. In this study, we address the question of causation and show that Pepd-null mice have increased antinuclear autoantibodies and raised serum IgA, accompanied by kidney immune complex deposition, consistent with a systemic lupus erythematosus-like disease. These features are associated with an accumulation of CD4 and CD8 effector T cells in the spleen and liver. Pepd deficiency leads to spontaneous T cell activation and proliferation into the effector subset, which is cell intrinsic and independent of Ag receptor specificity or antigenic stimulation. However, an increase in KLRG1+ effector CD8 cells is not observed in mixed chimeras, in which the autoimmune phenotype is also absent. Our findings link autoimmune susceptibility in PD to spontaneous T cell dysfunction, likely to be acting in combination with immune activators that lie outside the hemopoietic system but result from the abnormal metabolism or loss of nonenzymatic prolidase function. This knowledge provides insight into the role of prolidase in the maintenance of self-tolerance and highlights the importance of treatment to control T cell activation.

A Facile Method to Determine Prolidase Activity Using a Peptide-Specific Fluorometric Reaction

Prolidase is the only enzyme capable of cleaving imidodipeptides containing C-terminal proline (Pro) or hydroxyproline and plays a crucial role in several physiological processes such as wound healing and cell proliferation. Here, we developed a new method to determine prolidase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal glycine (Gly)-containing peptides using 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA can selectively react with Gly-Pro, the substrate for prolidase, and the prolidase activity is measured by monitoring the decrease in FL intensities. The prolidase activities in fibroblasts and HeLa cells were successfully measured by the proposed method. Compared with classical Chinard's method, our method does not require any caustic acids, pre-incubation to activate the enzyme, and heating for reaction with the detection reagent. The proposed method enables facile and specific measurement for biogenic prolidase activity.

Identification of peptides in Qingke baijiu and evaluation of its angiotensin converting enzyme (ACE) inhibitory activity and stability

The active peptides in Qingke baijiu fermented from Qingke (highland barley) are rarely reported. This work was designed to accurately identify peptides in Qingke baijiu and evaluate their angiotensin-converting enzyme inhibitory activities in vitro. Four novel peptides, Val-Val-Thr-Gly-Val-Gly-Gly-Gln (VVTGVGGQ), Leu-Pro-Val-Gly-Pro (LPVGP), Leu-Leu-Ser-Pro-Pro (LLSPP), and Phe-Pro-Leu-Gln-Pro-His-Gln-Pro (FPLQPHQP) were identified by Nano-UPLC-MS/MS. Molecular docking showed that LPVGP and FPLQPHQP had a high affinity with ACE (binding energy −8.78, −10.02 kcal mol−1), which matched its in vitro ACE inhibitory activity (IC50 9.05, 5.03 µM). This might be related to their high hydrophobicity. Moreover, three peptides have C-terminal proline, which may contribute to their anti-digestive activity. The content of LPVGP was over 91.23% after digestion, while the content of VVTGVGGQ dropped to 55.47%. Finally, the four peptides have no obvious toxicity to Caco-2 cells. This study clarifies the ACE inhibitory activity and the structure–activity relationship of the four peptides identified in Qingke baijiu.

Alkaloid Biosynthetic Enzyme Generates Diastereomeric Pair via Two Distinct Mechanisms.

Ergopeptines constitute one of the representative classes of ergoline alkaloids and carry a tripeptide extension on the lysergic acid core. In the current study, we discovered and structurally characterized newly isolated ergopeptine-like compounds named lentopeptins from a filamentous fungus Aspergillus lentulus, a close relative of A. fumigatus. Interestingly, in lentopeptins, the common lysergic acid moiety of ergopeptines is replaced by a cinnamic acid moiety at the N-terminus of the peptide segment. Moreover, lentopeptins lack the C-terminal proline residue necessary for the spontaneous cyclization of the peptide extension. Herein, we report the atypical lentopeptin biosynthetic pathway identified through targeted deletion of the len cluster biosynthetic genes predicted from the genome sequence. Further in vitro characterizations of the thiolation-terminal condensation-like (T-CT) didomain of the nonribosomal peptide synthetase LenA and its site-specific mutants revealed the mechanism of peptide release via diketopiperazine formation, an activity previously unreported for CT domains. Most intriguingly, in vitro assays of the cytochrome P450 LenC illuminated the unique mechanisms to generate two diastereomeric products. Lentopeptin A forms via a stereospecific hydroxylation, followed by a spontaneous bicyclic lactam core formation, while lentopeptin B is produced through an initial dehydrogenation, followed by a bicyclic lactam core formation and stereospecific hydration. Our results showcase how nature exploits common biosynthetic enzymes to forge new complex natural products effectively (213/250).

Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B.

CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.

Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of β-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

A Novel UPLC-MS/MS Method Identifies Organ-Specific Dipeptide Profiles.

Background: Amino acids have a central role in cell metabolism, and intracellular changes contribute to the pathogenesis of various diseases, while the role and specific organ distribution of dipeptides is largely unknown. Method: We established a sensitive, rapid and reliable UPLC-MS/MS method for quantification of 36 dipeptides. Dipeptide patterns were analyzed in brown and white adipose tissues, brain, eye, heart, kidney, liver, lung, muscle, sciatic nerve, pancreas, spleen and thymus, serum and urine of C57BL/6N wildtype mice and related to the corresponding amino acid profiles. Results: A total of 30 out of the 36 investigated dipeptides were detected with organ-specific distribution patterns. Carnosine and anserine were most abundant in all organs, with the highest concentrations in muscles. In liver, Asp-Gln and Ala-Gln concentrations were high, in the spleen and thymus, Glu-Ser and Gly-Asp. In serum, dipeptide concentrations were several magnitudes lower than in organ tissues. In all organs, dipeptides with C-terminal proline (Gly-Pro and Leu-Pro) were present at higher concentrations than dipeptides with N-terminal proline (Pro-Gly and Pro-Leu). Organ-specific amino acid profiles were related to the dipeptide profile with several amino acid concentrations being related to the isomeric form of the dipeptides. Aspartate, histidine, proline and serine tissue concentrations correlated with dipeptide concentrations, when the amino acids were present at the C- but not at the N-terminus. Conclusion: Our multi-dipeptide quantification approach demonstrates organ-specific dipeptide distribution. This method allows us to understand more about the dipeptide metabolism in disease or in healthy state.

PROLIDASE: A Review from Discovery to its Role in Health and Disease.

Prolidase (peptidase D), encoded by the PEPD gene, is a ubiquitously expressed cytosolic metalloproteinase, the only enzyme capable of cleaving imidodipeptides containing C-terminal proline or hydroxyproline. Prolidase catalyzes the rate-limiting step during collagen recycling and is essential in protein metabolism, collagen turnover, and matrix remodeling. Prolidase, therefore plays a crucial role in several physiological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Accordingly, mutations leading to loss of prolidase catalytic activity result in prolidase deficiency a rare autosomal recessive metabolic disorder characterized by defective wound healing. In addition, alterations in prolidase enzyme activity have been documented in numerous pathological conditions, making prolidase a useful biochemical marker to measure disease severity. Furthermore, recent studies underscore the importance of a non-enzymatic role of prolidase in cell regulation and infectious disease. This review aims to provide comprehensive information on prolidase, from its discovery to its role in health and disease, while addressing the current knowledge gaps.

The Role of Kruppel‐like Factor 6 in Prolidase Regulation

Prolidase, also known as peptidase D (PEPD), is a hydrolase that cleaves dipeptides containing C-terminal proline or hydroxyproline. These dipeptides are generated largely during collagen turnover, therefore prolidase catalysis is an essential rate-limiting step during collagen biosynthesis. Consequently, prolidase plays a significant role in collagen metabolism, matrix remodeling, and wound healing. Defective wound healing is one of the hallmarks of Prolidase Deficiency, a rare autosomal recessive disorder characterized by the excretion of prolidase substrates in the urine. Although prolidase is implicated in wound healing, its molecular and cellular regulation remains understudied. Our initial in silico analysis of the PEPD promoter (PEPDpro) identified key regulatory elements upstream of the transcription start site. We identified Kruppel-like factor 6 (KLF6), a zinc-finger transcription factor associated with vascular injury, wound healing, and collagen metabolism. To study the interaction between KLF6 and prolidase, we amplified the promoter from the human genome and inserted it into a luciferase reporter construct. Our preliminary data demonstrate that KLF6 enhances PEPD promoter activity in a dose-dependent manner. Additionally, KLF6 is regulated by transforming growth factor β1 (TGFβ1), and our data illustrate that TGFβ1 drives PEPD promoter activity. These results will generate new knowledge on the molecular regulation of prolidase and aid in developing therapeutic approaches to regulate prolidase expression in various physiological and pathological conditions such as wound healing.

Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model

SummaryRetinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.

Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics.

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.

Ultrasensitive Detection of NOTCH1 c.7544_7545delCT Mutations in Chronic Lymphocytic Leukemia by Droplet Digital PCR Reveals High Frequency of Subclonal Mutations and Predicts Clinical Outcome in Cases with Trisomy 12

NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P=0.031). Mutational burdens <1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.

Abstract 5145: A new discovering motif on SOS1 PR domain clues Grb2-SOS1 association

Abstract Grb2 is an adaptor protein that recruits Ras-specific guanine nucleotide exchange factor (GEF), such as Son of sevenless 1 (SOS1), to the plasma membrane. SOS1 exchanges GDP by GTP, activating Ras. Grb2 consists of an SH2 domain in the middle and flanks nSH3/cSH3 domains at both ends. Both nSH3/cSH3 domains bind to SOS1’s C-terminal proline rich (PR) domain and have the preference for interacting with the PXXPXR motif. Our NMR data suggest that the arginine residue, followed by prolines in the PXXPXR motif, is responsible for the SH3-SOS1 association, yielding large chemical shift perturbations (CSPs) on the binding interface of SH3. The SOS1’s PR motif, PVPPPVPPRRRP, exhibits the strongest binding affinity to both SH3 domains. Using comprehensive techniques involving molecular dynamics (MD) simulation and NMR measurement, we uncover a new SOS1’s PR motifs, PKLPPKTYKREH, which shows strong binding to cSH3 but exhibits very weak affinity to nSH3. Unlike the arginine residue in the PXXPXR motif, the lysine residue in the PXXPPKXXKR motif induces weak interaction with cSH3. However, high affinity interaction is mainly derived from the hydrophobic interaction between the PXXP motif and cSH3, indicating the different binding modes of interaction. Our results suggest that Grb2 nSH3/cSH3 bind to SOS1 at distinct locations and provide atomic detailed models of Grb2-SOS1 association. Recent Grb2 targeted therapy reveals that peptide inhibitors, which interrupt Grb2-SOS1 interaction, effectively prevent the migration of breast cancer cell. The mechanism of Grb2-SOS1 interaction may enlighten the promising drug treatment development. Citation Format: Tsung-Jen Liao, Hyunbum Jang, Ruth Nussinov, David Fushman. A new discovering motif on SOS1 PR domain clues Grb2-SOS1 association [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5145.

Purification and charicterization of angiotensin I-converting enzyme (ACE) inhibitory peptides with specific structure X-Pro

This study aimed to specifically prepare angiotensin I-converting enzyme (ACE) inhibitory peptides rich in C-terminal proline from oyster proteins using chymotrypsin and proline-specific endopeptidases (PSEases). The hydrolysates were purified with Sephadex G25, Superdex™ 30 Increase 10/300 GL gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The ACE inhibitory IC50 of purified fraction (G1E1C1) was 0.032 ± 0.003 mg/mL. According to ESI–MS and ESI–MS/MS analyses, there were three novel ACE inhibitory peptides in G1E1C1 fraction. Their sequences were Ser-Ala-Pro, Ala-Met-Pro and Thr-Ser-Gly-Pro. Molecular docking of peptides to ACE was studied. Metal–acceptor interactions and conventional hydrogen bonds greatly promoted the stability of peptides to ACE interaction. Pyrrole ring of proline may lead to higher inhibitory activity of peptides. It easily formed a Pi–alkyl interaction with aromatic ring residues (His353, His387, His513, and Phe512). Pi interactions may promote the effect of peptides on ACE. Also, C atom adjacent to the N atom of the pyrrole ring easily formed a carbon hydrogen bond with Ala354. The research discovered three novel ACE inhibitory peptides and provided a method to obtain ACE inhibitory peptides with specific structure X-Pro. The research result played an important role in revealing the structure–activity relationship of ACE inhibitory peptides and designing novel peptides with enhanced biological activity.