RESUMO
Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.
Assuntos
Metabolismo dos Carboidratos , Glicosiltransferases/metabolismo , Espectrometria de Massas/métodos , Glicosídeos/metabolismoRESUMO
This paper describes how angle-dependent resonances from molded plasmonic crystals can be used to improve real-time biosensing. First, an inexpensive and massively parallel approach to create single-use, two-dimensional metal nanopyramidal gratings was developed. Second, although constant in bulk dielectric environments, the sensitivities (resonance wavelength shift and resonance width) of plasmonic crystals to adsorbed molecular layers of varying thickness were found to depend on incident excitation angle. Third, protein binding at dilute concentrations of protein was carried out at an angle that optimized the signal to noise of our plasmonic sensing platform. This angle-dependent sensitivity, which is intrinsic to grating-based sensors, is a critical parameter that can assist in maximizing signal to noise.
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Arachis/química , Dissacarídeos/metabolismo , Lectinas/metabolismo , Proteínas de Plantas/metabolismo , Ressonância de Plasmônio de Superfície/instrumentação , Dissacarídeos/química , Lectinas/análise , Microtecnologia , Proteínas de Plantas/análise , Ligação Proteica , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/economiaRESUMO
Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) is effective against prostate cancer (PCa), but all patients relapse eventually. Poor understanding of the underlying resistance mechanisms represents a key barrier to development of more effective RLT. We investigate the proteome and phosphoproteome in a mouse model of PCa to identify signaling adaptations triggered by PSMA RLT. Methods: Therapeutic efficacy of PSMA RLT was assessed by tumor volume measurements, time to progression, and survival in C4-2 or C4-2 TP53-/- tumor-bearing nonobese diabetic scid γ-mice. Two days after RLT, the proteome and phosphoproteome were analyzed by mass spectrometry. Results: PSMA RLT significantly improved disease control in a dose-dependent manner. Proteome and phosphoproteome datasets revealed activation of genotoxic stress response pathways, including deregulation of DNA damage/replication stress response, TP53, androgen receptor, phosphatidylinositol-3-kinase/AKT, and MYC signaling. C4-2 TP53-/- tumors were less sensitive to PSMA RLT than were parental counterparts, supporting a role for TP53 in mediating RLT responsiveness. Conclusion: We identified signaling alterations that may mediate resistance to PSMA RLT in a PCa mouse model. Our data enable the development of rational synergistic RLT-combination therapies to improve outcomes for PCa patients.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Próstata , Antígeno Prostático EspecíficoRESUMO
PURPOSE: Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor. EXPERIMENTAL DESIGN: RM1 cells expressing different levels of PSMA (PSMA-, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA- RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1-3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified. RESULTS: 177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups. CONCLUSIONS: In the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.See related commentary by Ravi Kumar and Hofman, p. 2774.
Assuntos
Antígenos de Superfície , Antígeno Prostático Específico , Animais , Antígenos de Superfície/metabolismo , Dano ao DNA , Ligantes , Masculino , Camundongos , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata , Células Tumorais CultivadasRESUMO
225Ac-PSMA-617 targeted-therapy has demonstrated efficacy in 75-85% of patients; however, responses are not durable. We aimed to establish translatable mouse models of disseminated prostate cancer (PCa) to evaluate effectiveness of 225Ac-PSMA-617 at various disease stages. Methods: C4-2, C4-2B, or 22Rv1 cells were injected into the left ventricle of male NSG mice. Disease progression was monitored using bioluminescence imaging (BLI). For treatment, mice were injected with 40 kBq 225Ac-PSMA-617 at one (early treatment cohort) or three weeks (late treatment cohort) post-inoculation. Treatment efficacy was monitored by BLI of whole-body tumor burden. Mice were sacrificed based on body conditioning score. Results: C4-2 cells yielded metastases in liver, lungs, spleen, stomach, bones, and brain - achieving a clinically relevant model of widespread metastatic disease. The disease burden in the early treatment cohort was stable over 27 weeks in 5/9 mice and progressive in 4/9 mice. These mice were sacrificed due to brain metastases. Median survival of the late treatment cohort was superior to controls (13 vs. 7 weeks; p<0.0001) but inferior to that in the early treatment cohort (13 vs. 27 weeks; p<0.001). Late cohort mice succumbed to extensive liver involvement. The 22Rv1 and C4-2B systemic models were not used for treatment due to high kidney metastatic burden or low take rate, respectively. Conclusion: C4-2 cells reproduced metastatic cancer spread most relevantly. Early treatment with 225Ac-PSMA-617 prevented liver metastases and led to significant survival benefit. Late treatment improved survival without reducing tumor burden in the liver, the main site of metastasis. The current findings suggest that early 225Ac-PSMA-617 intervention is more efficacious in the setting of widespread metastatic PCa.
Assuntos
Actínio/uso terapêutico , Dipeptídeos/uso terapêutico , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Compostos Radiofarmacêuticos/uso terapêutico , Partículas alfa/uso terapêutico , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estudos de Viabilidade , Humanos , Masculino , Camundongos , Antígeno Prostático EspecíficoRESUMO
A potent class of isoquinoline-based α-N-heterocyclic carboxaldehyde thiosemicarbazone (HCT) compounds has been rediscovered; based upon this scaffold, three series of antiproliferative agents were synthesized through iterative rounds of methylation and fluorination modifications, with anticancer activities being potentiated by physiologically relevant levels of copper. The lead compound, HCT-13, was highly potent against a panel of pancreatic, small cell lung carcinoma, prostate cancer, and leukemia models, with IC50 values in the low-to-mid nanomolar range. Density functional theory (DFT) calculations showed that fluorination at the 6-position of HCT-13 was beneficial for ligand-copper complex formation, stability, and ease of metal-center reduction. Through a chemical genomics screen, we identify DNA damage response/replication stress response (DDR/RSR) pathways, specifically those mediated by ataxia-telangiectasia and Rad3-related protein kinase (ATR), as potential compensatory mechanism(s) of action following HCT-13 treatment. We further show that the cytotoxicity of HCT-13 is copper-dependent, that it promotes mitochondrial electron transport chain (mtETC) dysfunction, induces production of reactive oxygen species (ROS), and selectively depletes guanosine nucleotide pools. Lastly, we identify metabolic hallmarks for therapeutic target stratification and demonstrate the in vivo efficacy of HCT-13 against aggressive models of acute leukemias in mice.
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The trend to inform personalized molecular radiotherapy with molecular imaging diagnostics, a concept referred to as theranostics, has transformed the field of nuclear medicine in recent years. The development of theranostic pairs comprising somatostatin receptor (SSTR)-targeting nuclear imaging probes and therapeutic agents for the treatment of patients with neuroendocrine tumors (NETs) has been a driving force behind this development. With the Neuroendocrine Tumor Therapy (NETTER-1) phase 3 trial reporting encouraging results in the treatment of well-differentiated, metastatic midgut NETs, peptide radioligand therapy (RLT) with the 177Lu-labeled somatostatin analog (SSA) [177Lu]Lu-DOTA-TATE is now anticipated to become the standard of care. On the diagnostics side, the field is currently dominated by 68Ga-labeled SSAs for the molecular imaging of NETs with positron emission tomography-computed tomography (PET/CT). PET/CT imaging with SSAs such as [68Ga]Ga-DOTA-TATE, [68Ga]Ga-DOTA-TOC, and [68Ga]Ga-DOTA-NOC allows for NET staging with high accuracy and is used to qualify patients for RLT. Driven by the demand for PET/CT imaging of NETs, a commercial kit for the production of [68Ga]Ga-DOTA-TATE (NETSPOT) was approved by the U.S. Food and Drug Administration (FDA). The synthesis of 68Ga-labeled SSAs from a 68Ge/68Ga-generator is straightforward and allows for a decentralized production, but there are economic and logistic difficulties associated with these approaches that warrant the search for a viable, generator-independent alternative. The clinical introduction of an 18F-labeled SSTR-imaging probe can help mitigate the shortcomings of the generator-based synthesis approach, but despite extensive research efforts, none of the proposed 18F-labeled SSAs has been translated past prospective first-in-humans studies so far. Here, we review the current state of probe-development from a translational viewpoint and make a case for a clinically viable, 18F-labeled alternative to the current standard [68Ga]Ga-DOTA-TATE.
Assuntos
Antineoplásicos/administração & dosagem , Radioisótopos de Flúor/administração & dosagem , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/terapia , Somatostatina/análogos & derivados , Somatostatina/administração & dosagem , Nanomedicina Teranóstica/métodos , Pesquisa Biomédica/tendências , Hormônios/administração & dosagem , Humanos , Imagem Molecular/métodos , Terapia de Alvo Molecular/métodos , Estudos ProspectivosRESUMO
The development of a miniaturized sensing platform for the selective detection of chemical odorants could stimulate exciting scientific and technological opportunities. Oligopeptides are robust substrates for the selective recognition of a variety of chemical and biological species. Likewise, semiconducting nanowires are extremely sensitive gas sensors. Here we explore the possibilities and chemistries of linking peptides to silicon nanowire sensors for the selective detection of small molecules. The silica surface of the nanowires is passivated with peptides using amide coupling chemistry. The peptide/nanowire sensors can be designed, through the peptide sequence, to exhibit orthogonal responses to acetic acid and ammonia vapors, and can detect traces of these gases from "chemically camouflaged" mixtures. Through both theory and experiment, we find that this sensing selectivity arises from both acid/base reactivity and from molecular structure. These results provide a model platform for what can be achieved in terms of selective and sensitive "electronic noses."
Assuntos
Ácido Acético/análise , Amônia/análise , Técnicas Analíticas Microfluídicas/métodos , Nanofios/química , Oligopeptídeos/química , Ácido Acético/química , Amônia/química , Técnicas Biossensoriais/métodos , Modelos Químicos , Silício/químicaRESUMO
BACKGROUND: Prostate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) is a promising yet not curative approach in castration-resistant (CR) prostate cancer (PC). Rational combination therapies may improve treatment efficacy. Here, we explored the effect of androgen receptor blockade (ARB) on PSMA expression visualized by PET and its potential additive effect when combined with 177Lu-PSMA RLT in a mouse model of prostate cancer. METHODS: Mice bearing human CRPC (C4-2 cells) xenografts were treated with 10 mg/kg enzalutamide (ENZ), with 50 mg/kg bicalutamide (BIC), or vehicle (control) for 21 days. PSMA expression was evaluated by 68Ga-PSMA11 PET/CT and quantified by flow cytometry of tumor fine needle aspirations before treatment and on days 23, 29, 34, and 39 post-therapy induction. For the RLT combination approach, mice bearing C4-2 tumors were treated with 10 mg/kg ENZ or vehicle for 21 days before receiving either 15 MBq (84 GBq/µmol) 177Lu-PSMA617 or vehicle. DNA damage was assessed as phospho-γH2A.X foci in tumor biopsies. Reduction of tumor volume on CT and survival were used as study endpoints. RESULTS: Tumor growth was delayed by ARB while 68Ga-PSMA11 uptake increased up to 2.3-fold over time when compared to controls. ABR-induced upregulation of PSMA expression was confirmed by flow cytometry. Phospho-γH2A.X levels increased 1.8- and 3.4-fold at 48 h in response to single treatment ENZ or RLT and ENZ+RLT, respectively. Despite significantly greater DNA damage and persistent increase of PSMA expression at the time of RLT, no additional tumor growth retardation was observed in the ENZ+RLT group (vs. RLT only, p = 0.372 at day 81). Median survival did not improve significantly when ENZ was combined with RLT. CONCLUSION: ARB-mediated increases in PSMA expression in PC xenografts were evident by 68Ga-PSMA11 PET imaging and flow cytometry. 177Lu-PSMA617 effectively decreased C4-2 tumor size. However, while pre-treatment with ARB increased DNA damage significantly, it did not result in synergistic effects when combined with RLT.
RESUMO
BACKGROUND: Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced. METHODS: Both [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE. RESULTS: The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50⯱â¯6% (nâ¯=â¯15), overall decay-corrected radiochemical yield of 16⯱â¯1% (nâ¯=â¯5) in ~40â¯min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26â¯×â¯10-2â¯mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds. CONCLUSIONS: The combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.
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Compostos de Boro/química , Radioisótopos de Flúor , Dispositivos Lab-On-A-Chip , Octreotida/análogos & derivados , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioquímica/instrumentação , Receptores de Somatostatina/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Octreotida/química , Traçadores Radioativos , RadiometriaRESUMO
To improve prostate-specific membrane antigen (PSMA)-targeted theranostic approaches, robust murine models of prostate cancer are needed. However, important characteristics of preclinical PSMA imaging-that is, the reproducibility of the imaging signal and the relationship between quantitative cell surface PSMA expression and lesion detectability with small-animal PET/CT-have not been defined yet. Methods: Murine prostate cancer RM1 sublines (ras myc transformed cells of C57BL/6 prostate origin) expressing varying levels of human PSMA were injected into the shoulder of C57BL/6 mice on day 0. 68Ga-PSMA11 PET/CT was performed on days 7 and 8 and interpreted by 2 masked readers to determine interday and interreader reproducibility. PSMA expression was quantified on days 7 and 8 by flow cytometry of fine-needle aspiration tumor biopsy samples. Cell surface PSMA expression was correlated with PET signal. The threshold for PET positivity was based on the clinical Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE) criteria. Results: The maximum and average percentages of injected 68Ga-PSMA11 activity per gram of tissue (%IA/g) correlated nearly perfectly as determined by 2 independent readers and on 2 separate days (intraclass correlation coefficient, 1.00/0.89 and 0.95/0.88, respectively). The number of PSMA molecules per cell increased from the RM1-yellow fluorescent protein subline (PSMA-; 2,000/cell) to the RM1-low subline (PSMA+; 17,000/cell), the RM1-medium subline (PSMA++; 22,000/cell), and the RM1-PGLS subline (PSMA-positive, green fluorescent protein-positive, and luciferase-positive; PSMA+++; 45,000/cell). Expression levels correlated with the visual positivity rate on 68Ga-PSMA11 PET and with the PSMA PET %IA/g. The PSMA threshold for PET positivity was approximately 20,000 per cell. Signal correlation was close at lower PSMA levels (RM1-low to RM1-medium; 10-23 %IA/g) but was lost at higher PSMA levels (RM1-medium to RM1-PGLS; 23-27 %IA/g). Conclusion: The in vivo relationship between 68Ga-PSMA11 PET/CT and PSMA expression level in a murine model of prostate cancer was robust for lower cell surface PSMA expression levels (≤22,000/cell). Thus, preclinical 68Ga-PSMA11 PET/CT can be used as an imaging biomarker to test PSMA-targeted interventions in murine models.
Assuntos
Ácido Edético/análogos & derivados , Oligopeptídeos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata/diagnóstico por imagem , Animais , Transporte Biológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ácido Edético/metabolismo , Isótopos de Gálio , Radioisótopos de Gálio , Regulação Neoplásica da Expressão Gênica , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reprodutibilidade dos TestesRESUMO
Clinical 177Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for 177Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 × 106) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of 177Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo γ-counter measurement. DNA double-strand breaks were measured using anti-gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and 18F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios ± SEM were 19 ± 5, 10 ± 5, and 2 ± 0 for high, intermediate, and low (each n = 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks (≥5% γ-H2A.X-positive cells). High when compared with intermediate or low specific activity resulted in a lower mean ± SEM tumor load by histopathology (vital tissue, 4 ± 2 vs. 8 ± 3 mm2; n = 3 vs. 6), day-4 18F-FDG PET (metabolic volume, 87 ± 23 vs. 118 ± 14 mm3; n = 6 vs. 12), and day-7 CT (volume, 323 ± 122 vs. 590 ± 46 mm3; n = 3 vs. 6; P = 0.039). 177Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P = 0.021, n = 5/group) without subacute hematologic toxicity (n = 3/group). Conclusion:177Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.
Assuntos
Dipeptídeos/uso terapêutico , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Neoplasias da Próstata/radioterapia , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dipeptídeos/efeitos adversos , Dipeptídeos/farmacocinética , Fluordesoxiglucose F18 , Compostos Heterocíclicos com 1 Anel/efeitos adversos , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Imuno-Histoquímica , Lutécio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico por imagem , Compostos Radiofarmacêuticos/efeitos adversos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Carga TumoralRESUMO
Epigenetic regulation is directed, in part, by the correlated placement of histone post-translational modifications, but the mechanisms controlling correlated modifications are incompletely understood. Correlations arise from crosstalk among modifications and are frequently attributed to protein-protein interactions that recruit enzymes to existing histone modifications. Here we report the use of a peptide array to discover acetyltransferase-mediated crosstalks. We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate. Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation. Mutagenesis experiments demonstrate that PCAF specifically interprets H3 Arg8 modifications through interaction with residue Tyr640 on the surface of its catalytic domain, and this interaction regulates Lys14 acetylation by substrate discrimination. PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications. Together, this work describes a method for systematically mapping crosstalks and illustrates its application to the discovery and elucidation of novel PCAF crosstalks.