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Rational Design of Stapled Covalent Peptide Modifiers of Oncoprotein E6 from Human Papillomavirus.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-10 DOI: 10.1021/acschembio.4c00878
Cole Emanuelson, Yuta Naro, Olivia Shade, Melinda Liu, Sagar D Khare, Alexander Deiters

Human Papillomavirus (HPV) is linked to multiple cancers, most significantly cervical cancer, for which HPV infection is associated with nearly all cases. Essential to the oncogenesis of HPV is the function of the viral protein E6 and its role in degrading the cell cycle regulator p53. Degradation of p53, and the resultant loss of cell cycle control, is mediated by E6 recruitment of the E3 ubiquitin ligase E6AP and subsequent ubiquitination of p53. Here, we report the design of a stapled peptide that mimics the LxxLL α-helical domain of E6AP to bind and covalently label a cysteine residue specific to HPV-16 E6. Several acrylamide- and haloacetamide-based warheads were evaluated for reactivity and specificity, and a panel of hydrocarbon-stapled peptides was evaluated for enhanced binding affinity and increased proteolytic stability. Structure-based modeling was used to rationalize the observed trends in the reactivity of the warheads and the impact of the hydrocarbon staple position on the binding affinity of the stapled peptides. The development of a proteolytically stable and reactive peptide represents a new class of peptide-based inhibitors of protein-protein interactions with a potential therapeutic value toward HPV-derived cancers.

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引用次数: 0
G-Quadruplex-Based Splice Switching as a Therapeutic Approach in Duchenne Muscular Dystrophy.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-03 DOI: 10.1021/acschembio.4c00805
Ryo Iwase, Taro Ishiguro, Rintaro I Hara, Tetsuya Nagata, Takanori Yokota

RNA guanine (G)-quadruplexes (rG4) are unique noncanonical structures composed of stacked guanine quadruplexes that play diverse roles in regulating gene expression, from transcription to protein synthesis. This study proposes a new splice-switching therapy using G-quadruplex-inducing antisense oligonucleotides (G-ASOs) to reinstate dystrophin expression in Duchenne muscular dystrophy (DMD) models. G-ASOs consist of two functionally independent domains that enable the formation of RNA/DNA hetero-G-quadruplex (hG4) structures. The antisense domain binds to complementary sequences within the target RNA, while the G-rich domain, which contains a sequence of continuous guanines (G-tract), interacts with the G-rich region of target RNA to form an hG4 structure. This precise binding forms an hG4 structure that effectively interrupts alternative splicing. In contrast to the traditional methods that block sterically, this technique employs steric hindrance by forming hG4 structures. Significantly, our findings show that hG4 structures can still form effectively even when the G-rich region of the target RNA and the antisense sequence are as much as 70 nucleotides apart. To address the challenges associated with G-quadruplex formation via G-ASO self-assembly, we developed bulge-containing G-ASOs. This enhancement improves both the efficiency of hG4 formation and the induction of exon-skipping therapy. In summary, this study highlights the potential of G-ASOs in gene therapy, specifically DMD, and marks significant progress in the development of novel therapeutic strategies. These findings highlight the effectiveness of G-ASOs in exon-skipping therapy and demonstrate the advancements in RNA structural manipulation.

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引用次数: 0
Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-13 DOI: 10.1021/acschembio.4c00803
Andrew C D Lemmex, Jeremy Allred, Jason Ostergard, Jake Trask, Hannah N Bui, Michael J M Anderson, Benjamin Kopp, Oakley Streeter, Adam T Smiley, Natalia S Babilonia-Díaz, Bruce R Blazar, LeeAnn Higgins, Peter M Gordon, Joseph M Muretta, Wendy R Gordon

Notch plays critical roles in developmental processes and disease pathogenesis, which have led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling derived from a synthetic phage-display library targeting the Notch negative regulatory region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay with an IC50 of about 5 μM and in a Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19 μM affinity for the Notch NRR. We addressed the low affinity by fusion to a mutant varient of the β-pore-forming toxin aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with efficacy similar to that of other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

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引用次数: 0
Reengineering of Circularly Permuted Caspase-2 to Enhance Enzyme Stability and Enable Crystallographic Studies.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 DOI: 10.1021/acschembio.4c00795
Jessica L Fuller, Ke Shi, Steffen Pockes, Barry C Finzel, Karen H Ashe, Michael A Walters

Caspase activation has been linked to several diseases, including cancer, neurodegeneration, and inflammatory conditions, generating interest in targeting this family of proteases for drug development. Caspase-2 (Casp2) in particular has been implicated in Alzheimer's Disease (AD) by cleaving tau protein into fragment Δtau314, which reversibly impairs cognitive and synaptic function. Thus, Casp2 inhibition could be a useful strategy for therapeutic treatment of AD. To that end, we have previously synthesized and characterized various series of peptide and peptidomimetic inhibitors that demonstrate potency and selectivity for Casp2 over caspase-3 (Casp3). Despite promising developments in the design of selective Casp2 inhibitors, low expression yields of Casp2 hinder crystallographic experiments and make structure-based design challenging. The design of circularly permuted (cp) Casp2 increased protein yields considerably; however, this protein could not be crystallized. This article describes the characterization of ten novel cpCasp2 mutants, designed with the goal of increasing stability and facilitating crystallization. Gratifyingly, engineered mutant JF1cpCasp2 displayed high relative stability and was readily crystallizable with the canonical Casp2 inhibitor AcVDVAD-CHO, leading to what we believe to be the first crystal structures of any reverse caspase in the PDB. Moreover, we have reported the structure of JF1cpCasp2 with our recently described Casp2-selective inhibitor MUR-65, which revealed a unique interaction with Arg417 in the binding pocket. Overall, JF1cpCasp2 has proven valuable for structure-based design and expanding understanding of Casp2 inhibition, with potential implications for drug discovery and the development of more selective compounds.

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引用次数: 0
Bisecting N-Acetylglucosamine of the N-Glycan of Immunoglobulin G Does Not Affect Binding to Fc Gamma Receptors.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-02-19 DOI: 10.1021/acschembio.4c00807
Gerlof P Bosman, Inèz D Stoof, Hans P Bastiaansen, Linda Quarles van Ufford, Justyna M Dobruchowska, Jan-Willem H Langenbach, Bhargavi M Boruah, Kelley W Moremen, Arthur E H Bentlage, Suzanne N Lissenberg-Thunnissen, Gestur Vidarsson, Geert-Jan Boons

Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-N-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior in vitro and in vivo activities. The N-glycan of the Fc-region of these mAbs have increased levels of bisecting N-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the N-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc2Man3GlcNAc2) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.

{"title":"Bisecting <i>N</i>-Acetylglucosamine of the <i>N</i>-Glycan of Immunoglobulin G Does Not Affect Binding to Fc Gamma Receptors.","authors":"Gerlof P Bosman, Inèz D Stoof, Hans P Bastiaansen, Linda Quarles van Ufford, Justyna M Dobruchowska, Jan-Willem H Langenbach, Bhargavi M Boruah, Kelley W Moremen, Arthur E H Bentlage, Suzanne N Lissenberg-Thunnissen, Gestur Vidarsson, Geert-Jan Boons","doi":"10.1021/acschembio.4c00807","DOIUrl":"10.1021/acschembio.4c00807","url":null,"abstract":"<p><p>Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-<i>N</i>-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior <i>in vitro</i> and <i>in vivo</i> activities. The <i>N</i>-glycan of the Fc-region of these mAbs have increased levels of bisecting <i>N</i>-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the <i>N</i>-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc<sub>2</sub>Man<sub>3</sub>GlcNAc<sub>2</sub>) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"680-689"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934091/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-07 DOI: 10.1021/acschembio.4c00783
Gwen Tjallinks, Nicolò Angeleri, Quoc-Thai Nguyen, Barbara Mannucci, Mark Arentshorst, Jaap Visser, Arthur F J Ram, Marco W Fraaije, Andrea Mattevi

Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.

{"title":"Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis.","authors":"Gwen Tjallinks, Nicolò Angeleri, Quoc-Thai Nguyen, Barbara Mannucci, Mark Arentshorst, Jaap Visser, Arthur F J Ram, Marco W Fraaije, Andrea Mattevi","doi":"10.1021/acschembio.4c00783","DOIUrl":"10.1021/acschembio.4c00783","url":null,"abstract":"<p><p>Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from <i>Penicillium chrysogenum</i> with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its <i>out</i> state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from <i>P. chrysogenum</i> in <i>Aspergillus niger</i> with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"646-655"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Glycated α-Synuclein Renders Glial Cell Activation and Induces Degeneration of Dopaminergic Neurons: A Potential Implication for the Development of Parkinson's Disease.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-02-21 DOI: 10.1021/acschembio.4c00777
Sayan Chatterjee, Arvind Verma, Harsh Thakkar, Ravi P Shah, Amit Khairnar

Accumulation of misfolded α-synuclein (α-Syn) leads to the formation of Lewy bodies and is a major hallmark of Parkinson's disease (PD). The accumulation of α-Syn involves several post-translational modifications. Recently, though, glycation of α-Syn (advanced glycation end products) and activation of the receptor for advanced glycation end products (RAGE) have been linked to neuroinflammation, which leads to oxidative stress and accumulation of α-Syn. The present study aims to detect the effect of glycated α-Syn (gly-α-Syn)-induced synucleinopathy and loss of dopaminergic (DAergic) neurons in the development of PD. We isolated, purified, and prepared glycated recombinant human α-Syn using d-ribose. Gly-α-Syn was characterized by SDS-PAGE, intact mass analysis, and bottom-up peptide sequence through LC-HRMS/MS. The aggregation propensity of gly-α-Syn has been verified by morphological and shape analysis through Bio-AFM. The gly-α-Syn (2 μg/μL) was injected stereotaxically in the substantia nigra (SN) of ICR mice (3-4 months) and compared with the normal α-Syn, d ribose, and Tris-HCl/artificial CSF groups. 56 days postsurgery (DPS), an immunohistochemical examination was conducted to investigate gly-α-Syn-induced α-Syn accumulation, neuroinflammation, and neurodegeneration. The glycation of α-Syn led to the expression of transglutaminase 2 (TGM2), an enzyme that cross-linked with AGEs and may have caused the accumulation of α-Syn. Significant RAGE activation was also observed in gly-α-Syn, which might have induced glial cell activation, resulting in oxidative stress and, ultimately, apoptosis of dopaminergic neurons. It is important to note that TGM2, phosphorylated α-Syn, RAGE expression, and glial cell activation were only found in the gly-α-Syn group and not in the other groups. This suggests that gly-α-Syn plays a major role in synucleinopathy, neuroinflammation, and neurodegeneration. Overall, the present study demonstrated glycation of α-Syn as one of the important age-associated post-translational modifications that are involved in the degeneration of dopaminergic neurons, at least in a subset of the diabetic patients susceptible to developing PD.

{"title":"Glycated α-Synuclein Renders Glial Cell Activation and Induces Degeneration of Dopaminergic Neurons: A Potential Implication for the Development of Parkinson's Disease.","authors":"Sayan Chatterjee, Arvind Verma, Harsh Thakkar, Ravi P Shah, Amit Khairnar","doi":"10.1021/acschembio.4c00777","DOIUrl":"10.1021/acschembio.4c00777","url":null,"abstract":"<p><p>Accumulation of misfolded α-synuclein (α-Syn) leads to the formation of Lewy bodies and is a major hallmark of Parkinson's disease (PD). The accumulation of α-Syn involves several post-translational modifications. Recently, though, glycation of α-Syn (advanced glycation end products) and activation of the receptor for advanced glycation end products (RAGE) have been linked to neuroinflammation, which leads to oxidative stress and accumulation of α-Syn. The present study aims to detect the effect of glycated α-Syn (gly-α-Syn)-induced synucleinopathy and loss of dopaminergic (DAergic) neurons in the development of PD. We isolated, purified, and prepared glycated recombinant human α-Syn using d-ribose. Gly-α-Syn was characterized by SDS-PAGE, intact mass analysis, and bottom-up peptide sequence through LC-HRMS/MS. The aggregation propensity of gly-α-Syn has been verified by morphological and shape analysis through Bio-AFM. The gly-α-Syn (2 μg/μL) was injected stereotaxically in the substantia nigra (SN) of ICR mice (3-4 months) and compared with the normal α-Syn, d ribose, and Tris-HCl/artificial CSF groups. 56 days postsurgery (DPS), an immunohistochemical examination was conducted to investigate gly-α-Syn-induced α-Syn accumulation, neuroinflammation, and neurodegeneration. The glycation of α-Syn led to the expression of transglutaminase 2 (TGM2), an enzyme that cross-linked with AGEs and may have caused the accumulation of α-Syn. Significant RAGE activation was also observed in gly-α-Syn, which might have induced glial cell activation, resulting in oxidative stress and, ultimately, apoptosis of dopaminergic neurons. It is important to note that TGM2, phosphorylated α-Syn, RAGE expression, and glial cell activation were only found in the gly-α-Syn group and not in the other groups. This suggests that gly-α-Syn plays a major role in synucleinopathy, neuroinflammation, and neurodegeneration. Overall, the present study demonstrated glycation of α-Syn as one of the important age-associated post-translational modifications that are involved in the degeneration of dopaminergic neurons, at least in a subset of the diabetic patients susceptible to developing PD.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"632-645"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of RufT Thioesterase Domain Reveals Insights into Rufomycin Cyclization and the Biosynthetic Origin of Rufomyazine.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-06 DOI: 10.1021/acschembio.4c00802
Yaoyu Ding, Gustavo Perez-Ortiz, Alexandra-Georgiana Butulan, Hamzah Sharif, Sarah M Barry

The nonribosomal cyclic peptides (NRcPs) rufomycins, produced by Streptomyces atratus, have attracted attention as antimycobacterials. Thus, there has been interest in engineering the corresponding biosynthetic pathway to produce novel derivatives. We have thus investigated the type I thioesterase (TE) of the NRPS RufT that catalyzes rufomycin peptide macrocyclization to understand its tolerance to changes in substrate peptide sequence. In contrast to our previously reported efficient cyclization chemistry, the recombinant RufT-TE domain and RufT-PCP-TE didomain, while tolerating some substrate structural changes, both produce high levels of hydrolyzed peptide. Closer analysis led to the identification of the natural product diketopiperazine rufomyazine in assays. The data indicate, with significant implications for rufomycin production, that RufT produces both cyclic and linear peptides. We propose that rufomyazine forms non-enzymatically from the linear peptide. In addition, it provides evidence for TE domains as gatekeepers in NRPS biosynthesis.

{"title":"Characterization of RufT Thioesterase Domain Reveals Insights into Rufomycin Cyclization and the Biosynthetic Origin of Rufomyazine.","authors":"Yaoyu Ding, Gustavo Perez-Ortiz, Alexandra-Georgiana Butulan, Hamzah Sharif, Sarah M Barry","doi":"10.1021/acschembio.4c00802","DOIUrl":"10.1021/acschembio.4c00802","url":null,"abstract":"<p><p>The nonribosomal cyclic peptides (NRcPs) rufomycins, produced by <i>Streptomyces atratus</i>, have attracted attention as antimycobacterials. Thus, there has been interest in engineering the corresponding biosynthetic pathway to produce novel derivatives. We have thus investigated the type I thioesterase (TE) of the NRPS RufT that catalyzes rufomycin peptide macrocyclization to understand its tolerance to changes in substrate peptide sequence. In contrast to our previously reported efficient cyclization chemistry, the recombinant RufT-TE domain and RufT-PCP-TE didomain, while tolerating some substrate structural changes, both produce high levels of hydrolyzed peptide. Closer analysis led to the identification of the natural product diketopiperazine rufomyazine in assays. The data indicate, with significant implications for rufomycin production, that RufT produces both cyclic and linear peptides. We propose that rufomyazine forms non-enzymatically from the linear peptide. In addition, it provides evidence for TE domains as gatekeepers in NRPS biosynthesis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"573-580"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934086/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Imaging and Tracking RNA in Live Mammalian Cells via Fluorogenic Photoaffinity Labeling.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-02-15 DOI: 10.1021/acschembio.4c00848
Alexandria L Quillin, Diane B Karloff, Tewoderos M Ayele, Tatiana F Flores, Gerry Chen, Zachary T McEachin, Arielle N Valdez-Sinon, Jennifer M Heemstra

Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the inherently temporary nature of the noncovalent aptamer-fluorogen interaction limits the utility of these systems in that imaging does not withstand dye washout, and dye dissociation can compromise RNA tracking. We propose that these limitations can be averted through covalent RNA labeling. Here, we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable diazirine reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to achieve spatiotemporal control over RNA labeling. To demonstrate this approach, we incorporated a photoaffinity linker into malachite green and fused a single copy of the malachite green aptamer to a Cajal body-associated small nuclear RNA of interest as well as a cytoplasmic mRNA. We observed improved sensitivity for live cell imaging of the target RNA upon UV irradiation and demonstrated visualization of RNA dynamics over a time scale of minutes. The covalent attachment uniquely enables these time-resolved experiments, whereas in noncovalent approaches, the dye molecule can be transferred between different RNA molecules, compromising tracking. We envision future applications of this method for a wide range of investigations into the cellular localization, dynamics, and protein-binding properties of cellular RNAs.

{"title":"Imaging and Tracking RNA in Live Mammalian Cells via Fluorogenic Photoaffinity Labeling.","authors":"Alexandria L Quillin, Diane B Karloff, Tewoderos M Ayele, Tatiana F Flores, Gerry Chen, Zachary T McEachin, Arielle N Valdez-Sinon, Jennifer M Heemstra","doi":"10.1021/acschembio.4c00848","DOIUrl":"10.1021/acschembio.4c00848","url":null,"abstract":"<p><p>Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the inherently temporary nature of the noncovalent aptamer-fluorogen interaction limits the utility of these systems in that imaging does not withstand dye washout, and dye dissociation can compromise RNA tracking. We propose that these limitations can be averted through covalent RNA labeling. Here, we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable diazirine reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to achieve spatiotemporal control over RNA labeling. To demonstrate this approach, we incorporated a photoaffinity linker into malachite green and fused a single copy of the malachite green aptamer to a Cajal body-associated small nuclear RNA of interest as well as a cytoplasmic mRNA. We observed improved sensitivity for live cell imaging of the target RNA upon UV irradiation and demonstrated visualization of RNA dynamics over a time scale of minutes. The covalent attachment uniquely enables these time-resolved experiments, whereas in noncovalent approaches, the dye molecule can be transferred between different RNA molecules, compromising tracking. We envision future applications of this method for a wide range of investigations into the cellular localization, dynamics, and protein-binding properties of cellular RNAs.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"707-720"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11952673/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In Situ Labeling of Pathogenic Tau Using Photo-Affinity Chemical Probes. 利用光亲和性化学探针对致病性 Tau 进行原位标记
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-03-13 DOI: 10.1021/acschembio.5c00073
Pengju Nie, You Wu, John Robinson, Shekar Mekala, Virginia M Y Lee, Yue-Ming Li

Tau aggregation plays a crucial role in the development of Alzheimer's disease (AD). Developing specific techniques that can isolate pathogenic tau from brain tissue is important for understanding tauopathies and advancing targeted therapies. Here, we develop photoaffinity small molecular probes and a novel method for in situ tissue labeling and investigate their activity in interacting with tau in cells and AD patient brains. Based on the reported chemical structures of tau PET tracers, we designed and synthesized two tau-specific probes, namely, Tau-2 and Tau-4. After validation in cell, mouse model, and patient brain samples, our photolabeling results suggested that Tau-2 effectively labels soluble tau in cell and mouse models, while Tau-4 selectively binds high-molecular-weight tau aggregates in late-stage AD patient brain tissues. Proteomic analysis verified the specific isolation of pathogenic tau from AD brain samples. Collectively, these findings underscore the potential of our photoaffinity probes as powerful tools for investigating tau proteins and neurofibrillary tangles in neurodegenerative diseases.

{"title":"<i>In Situ</i> Labeling of Pathogenic Tau Using Photo-Affinity Chemical Probes.","authors":"Pengju Nie, You Wu, John Robinson, Shekar Mekala, Virginia M Y Lee, Yue-Ming Li","doi":"10.1021/acschembio.5c00073","DOIUrl":"10.1021/acschembio.5c00073","url":null,"abstract":"<p><p>Tau aggregation plays a crucial role in the development of Alzheimer's disease (AD). Developing specific techniques that can isolate pathogenic tau from brain tissue is important for understanding tauopathies and advancing targeted therapies. Here, we develop photoaffinity small molecular probes and a novel method for <i>in situ</i> tissue labeling and investigate their activity in interacting with tau in cells and AD patient brains. Based on the reported chemical structures of tau PET tracers, we designed and synthesized two tau-specific probes, namely, Tau-2 and Tau-4. After validation in cell, mouse model, and patient brain samples, our photolabeling results suggested that Tau-2 effectively labels soluble tau in cell and mouse models, while Tau-4 selectively binds high-molecular-weight tau aggregates in late-stage AD patient brain tissues. Proteomic analysis verified the specific isolation of pathogenic tau from AD brain samples. Collectively, these findings underscore the potential of our photoaffinity probes as powerful tools for investigating tau proteins and neurofibrillary tangles in neurodegenerative diseases.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"581-591"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143622806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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