bims-cepepe 2024-06-09 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–06–09
nineteen papers selected by
Henry Lamb, Queensland University of Technology

Chemical synthesis of grafted cyclotides using a "plug and play" approach.
De novo design of peptides that bind specific conformers of α-synuclein.
De Novo Discovery of Pseudo-Natural Prenylated Macrocyclic Peptide Ligands.
Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.
Author Correction: A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.
Cyclic β2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.
Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.
Use of a head-to-tail peptide cyclase to prepare hybrid RiPPs.
Has the blood-brain barrier finally been busted?
A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.
Enhancing Targeted Drug Delivery through Cell-Specific Endosomal Escape.
Comparing Cell Penetration of Biotherapeutics across Human Cell Lines.
Recent advances in cell-based in vitro models for predicting drug permeability across brain, intestinal, and pulmonary barriers.
Synthesis and preclinical evaluation of 68Ga-labeled PSMA tracers with improved pharmacological properties.
Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.
Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation.
Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.
Bait and Cleave: Exosite-Binding Peptides on Quantum Dots Selectively Accelerate Protease Activity for Sensing with Enhanced Sensitivity.
Metastatic melanoma to the urinary bladder with synchronous or metachronous metastasis to other organs: Clinicopathological characteristics and outcome.

RSC Chem Biol. 2024 Jun 05. 5(6): 567-571

Chemical synthesis of grafted cyclotides using a "plug and play" approach.

Johannes Koehbach, Edin Muratspahić, Zakaria M Ahmed, Andrew M White, Nataša Tomašević, Thomas Durek, Richard J Clark, Christian W Gruber, David J Craik.

Cyclotides are a diverse class of plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology. Their remarkable structural stability and resistance to proteolytic degradation can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. Thus, cyclotides have emerged as powerful scaffold molecules for designing peptide-based therapeutics. The chemical engineering of cyclotides has generated novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. However key challenges potentially limit the widespread use of cyclotides in molecular grafting applications. Folding of cyclotides containing bioactive epitopes remains a major bottleneck in cyclotide synthesis. Here we present a modular 'plug and play' approach that effectively bypasses problems associated with the oxidative folding of cyclotides. By grafting onto a pre-formed acyclic cyclotide-like scaffold we show that difficult-to-graft sequences can be easily obtained and can target GPCRs with nanomolar affinities and potencies. We further show the suitability of this new method to graft other complex epitopes including structures with additional disulfide bonds that are not readily available via currently employed chemical methods, thus fully unlocking cyclotides to be used in drug design applications.

DOI: https://doi.org/10.1039/d4cb00008k
Chem Sci. 2024 Jun 05. 15(22): 8414-8421

De novo design of peptides that bind specific conformers of α-synuclein.

Hailey M Wallace, Hyunjun Yang, Sophia Tan, Henry S Pan, Rose Yang, Junyi Xu, Hyunil Jo, Carlo Condello, Nicholas F Polizzi, William F DeGrado.

Insoluble amyloids rich in cross-β fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloids in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aβ40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins.

DOI: https://doi.org/10.1039/d3sc06245g
Angew Chem Int Ed Engl. 2024 Jun 05. e202409973

De Novo Discovery of Pseudo-Natural Prenylated Macrocyclic Peptide Ligands.

Sumika Inoue, Dinh Thanh Nguyen, Keisuke Hamada, Rika Okuma, Chikako Okada, Masahiro Okada, Ikuro Abe, Toru Sengoku, Yuki Goto, Hiroaki Suga.

  Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.

Keywords:  in vitro selection; macrocyclic ligands; peptides; prenyltransferase; pseudo-natural peptides

DOI:  https://doi.org/10.1002/anie.202409973
Nucleic Acids Res. 2024 Jun 03. pii: gkae471. [Epub ahead of print]

Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.

Francesco Merlino, Simona Marzano, Pasquale Zizza, Federica D'Aria, Nicola Grasso, Alice Carachino, Sara Iachettini, Annamaria Biroccio, Silvia Di Fonzo, Paolo Grieco, Antonio Randazzo, Jussara Amato, Bruno Pagano.

Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.

DOI: https://doi.org/10.1093/nar/gkae471
Nat Commun. 2024 Jun 07. 15(1): 4868

Author Correction: A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.

Kevin B Reed, Sierra M Brooks, Jordan Wells, Kristin J Blake, Minye Zhao, Kira Placido, Simon d'Oelsnitz, Adit Trivedi, Shruti Gadhiyar, Hal S Alper.

DOI: https://doi.org/10.1038/s41467-024-49314-w
Bull Chem Soc Jpn. 2024 May;97(5): uoae018

Cyclic β2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Takashi Miura, Tika R Malla, Lennart Brewitz, Anthony Tumber, Eidarus Salah, Kang Ju Lee, Naohiro Terasaka, C David Owen, Claire Strain-Damerell, Petra Lukacik, Martin A Walsh, Akane Kawamura, Christopher J Schofield, Takayuki Katoh, Hiroaki Suga.

  Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

Keywords:  macrocyclic peptide; protease inhibition; β-amino acid

DOI:  https://doi.org/10.1093/bulcsj/uoae018
J Am Chem Soc. 2024 Jun 06.

Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.

Yaoyu Ding, Edward Lambden, Jessica Peate, Lewis J Picken, Thomas W Rees, Gustavo Perez-Ortiz, Sophie A Newgas, Lucy A R Spicer, Thomas Hicks, Jeannine Hess, Martin B Ulmschneider, Manuel M Müller, Sarah M Barry.

Nonribosomal cyclic peptides (NRcPs) are structurally complex natural products and a vital pool of therapeutics, particularly antibiotics. Their structural diversity arises from the ability of the multidomain enzyme assembly lines, nonribosomal peptide synthetases (NRPSs), to utilize bespoke nonproteinogenic amino acids, modify the linear peptide during elongation, and catalyze an array of cyclization modes, e.g., head to tail, side chain to tail. The study and drug development of NRcPs are often limited by a lack of easy synthetic access to NRcPs and their analogues, with selective macrolactamization being a major bottleneck. Herein, we report a generally applicable chemical macrocyclization method of unprecedented speed and selectivity. Inspired by biosynthetic cyclization, it combines the deprotected linear biosynthetic precursor peptide sequence with a highly reactive C-terminus to produce NRcPs and analogues in minutes. The method was applied to several NRcPs of varying sequences, ring sizes, and cyclization modes including rufomycin, colistin, and gramicidin S with comparable success. We thus demonstrate that the linear order of modules in NRPS enzymes that determines peptide sequence encodes the key structural information to produce peptides conformationally biased toward macrocyclization. To fully exploit this conformational bias synthetically, a highly reactive C-terminal acyl azide is also required, alongside carefully balanced pH and solvent conditions. This allows for consistent, facile cyclization of exceptional speed, selectivity, and atom efficiency. This exciting macrolactamization method represents a new enabling technology for the biosynthetic study of NRcPs and their development as therapeutics.

DOI: https://doi.org/10.1021/jacs.4c04711
Chem Commun (Camb). 2024 Jun 04.

Use of a head-to-tail peptide cyclase to prepare hybrid RiPPs.

Tung Le, Dongtianyu Zhang, Rachel M Martini, Subhanip Biswas, Wilfred A van der Donk.

Cyclotides and lanthipeptides are cyclic peptide natural products with promising bioengineering potential. No peptides have been isolated that contain both structural motifs defining these two families, an N-to-C cyclised backbone and lanthionine linkages. We combined their biosynthetic machineries to produce hybrid structures that possess improved activity or stability, demonstrate how the AEP-1 plant cyclase can be utilised to complete the maturation of the sactipeptide subtilosin A, and present head-to-tail cyclisation of the glycocin sublancin. These studies show the plasticity of AEP-1 and its utilisation alongside other post-translational modifications.

DOI: https://doi.org/10.1039/d3cc04919a
Oncologist. 2024 Jun 05. pii: oyae131. [Epub ahead of print]

Has the blood-brain barrier finally been busted?

David J Stewart.

DOI: https://doi.org/10.1093/oncolo/oyae131
Microb Cell Fact. 2024 Jun 05. 23(1): 166

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.

Martin Gibisch, Matthias Müller, Christopher Tauer, Bernd Albrecht, Rainer Hahn, Monika Cserjan-Puschmann, Gerald Striedner.

   BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.
RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.
CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

Keywords:  Aprotinin; CASPON™ tag; Fed-batch cultivation; Parathyroid hormone; Plectasin; Recombinant peptides; Somatostatin

DOI:  https://doi.org/10.1186/s12934-024-02446-6
ChemMedChem. 2024 Jun 03. e202400274

Enhancing Targeted Drug Delivery through Cell-Specific Endosomal Escape.

Pengwen Chen, Horacio Cabral.

  Endosome is a major barrier in the intracellular delivery of drugs, especially for biologics, such as proteins, peptides, and nucleic acids. After being endocytosed, these cargos will be trapped inside the endosomal compartments and finally degraded in the lysosomes. Thus, various strategies have been developed to facilitate the escape of cargos from the endosomes to improve the intracellular delivery efficiency. While the majority of the studies are focusing on strengthening the endosomal escape capability to maximize the delivery outcome, recent evidence suggests that a careful control of the endosomal escape process could provide opportunity for targeted drug delivery. In this concept review, we examined current delivery systems that can sense intra-endosomal factors or external stimuli for controlling endosomal escape toward a targeted intracellular delivery of cargos. Furthermore, the prospects and challenges of such strategies are discussed.

Keywords:  Biologics; Endosomal escape; Intracellular delivery; Nanomedicine; mRNA

DOI:  https://doi.org/10.1002/cmdc.202400274
ACS Chem Biol. 2024 Jun 05.

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines.

Nefeli Batistatou, Joshua A Kritzer.

A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

DOI: https://doi.org/10.1021/acschembio.4c00211
Expert Opin Drug Metab Toxicol. 2024 Jun 08.

Recent advances in cell-based in vitro models for predicting drug permeability across brain, intestinal, and pulmonary barriers.

Bassma Eltanameli, Janny Piñeiro-Llanes, Rodrigo Cristofoletti.

   INTRODUCTION: Recent years have witnessed remarkable progress in the development of cell-based in vitro models aimed at predicting drug permeability, particularly focusing on replicating the barrier properties of the blood-brain barrier (BBB), intestinal epithelium, and lung epithelium.
AREA COVERED: This review provides an overview of 2D in vitro platforms, including monocultures and co-culture systems, highlighting their respective advantages and limitations. Additionally, it discusses tools and techniques utilized to overcome these limitations, paving the way for more accurate predictions of drug permeability. Furthermore, this review delves into emerging technologies, particularly microphysiological systems (MPS), encompassing static platforms such as organoids and dynamic platforms like microfluidic devices. Literature searches were performed using PubMed and Google Scholar. We focus on key terms such as in vitro permeability models, MPS, organoids, intestine, BBB, and lungs.
EXPERT OPINION: The potential of these MPS to mimic physiological conditions more closely offers promising avenues for drug permeability assessment. However, transitioning these advanced models from bench to industry requires rigorous validation against regulatory standards. Thus, there is a pressing need to validate MPS to industry and regulatory agency standards to exploit their potential in drug permeability prediction fully. This review underscores the importance of such validation processes to facilitate the translation of these innovative technologies into routine pharmaceutical practice.

Keywords:  Absorption; Blood-brain barrier; IVIVE; Microfluidic systems; Organoids; Tight junction; Transepithelial electrical resistance; in vitro-in vivo extrapolation

DOI:  https://doi.org/10.1080/17425255.2024.2366390
Eur J Med Chem. 2024 May 28. pii: S0223-5234(24)00425-2. [Epub ahead of print]274 116545

Synthesis and preclinical evaluation of 68Ga-labeled PSMA tracers with improved pharmacological properties.

Haodong Hou, Yixiang Lin, Yuan Pan, Yuze Ma, Guihua Hou, Xiangyang Sun, Feng Gao.

  Prostate cancer (PCa) is one of the most common tumors in men, with the overexpression of prostate-specific membrane. In this study, we developed four new 68Ga-labeled PSMA-targeting tracers by introducing quinoline, phenylalanine and decanoic acid groups to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Four radiotracers were synthesized with radiochemical purity >95 %, and exhibited high stability in vivo and in vitro. The inhibition constants (Ki) of SDTWS01-04 to PSMA were in the nanomolar range (<10 nM). Micro PET/CT imaging and biodistribution analysis revealed that 68Ga-SDTWS01 enabled clear tumor visualization in PET images at 1.5 h post-injection, with excellent pharmacokinetic properties. Notably, the kidney uptake of 68Ga-SDTWS01 significantly reduced, with higher tumor-to-kidney ratio (0.36 ± 0.02), tumor-to-muscle ratio (24.31 ± 2.10), compared with 68Ga-PSMA-11 (T/K: 0.15 ± 0.01; T/M: 14.97 ± 1.40), suggesting that 68Ga-SDTWS01 is a promising radiotracer for the diagnosis of PCa. Moreover, SDTWS01 with a chelator DOTA could also label 177Lu and 225Ac, which could be used for the treatment of PCa.

Keywords:  (68)Ga; Labelling; Positron Emission Tomography (PET); Prostate-specific membrane antigen (PSMA); Radiotracer

DOI:  https://doi.org/10.1016/j.ejmech.2024.116545
Sci Rep. 2024 06 05. 14(1): 12935

Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

Debasis Sahu, Charu Gupta, Ragothaman M Yennamalli, Shikha Sharma, Saugata Roy, Sadaf Hasan, Pawan Gupta, Vishnu Kumar Sharma, Sujit Kashyap, Santosh Kumar, Ved Prakash Dwivedi, Xiangli Zhao, Amulya Kumar Panda, Hasi Rani Das, Chuan-Ju Liu.

  The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Keywords:  Collagen-induced arthritis (CIA); Inflammation; Nuclear factor kappa B (NFκB); Peptide; Tumor necrosis factor (TNF)α

DOI:  https://doi.org/10.1038/s41598-024-63790-6
PLoS One. 2024 ;19(6): e0303882

Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation.

Darwin Ferng, Wesley Sun, Bih-Hwa Shieh.

Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.

DOI: https://doi.org/10.1371/journal.pone.0303882
J Med Chem. 2024 Jun 06.

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

Florian Stockinger, Pascal Poc, Alexander Möhwald, Sandra Karch, Stephanie Häfner, Christian Alzheimer, Guillaume Sandoz, Tobias Huth, Johannes Broichhagen.

The prevailing but not undisputed amyloid cascade hypothesis places the β-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00339
ACS Nano. 2024 Jun 06.

Bait and Cleave: Exosite-Binding Peptides on Quantum Dots Selectively Accelerate Protease Activity for Sensing with Enhanced Sensitivity.

Katherine D Krause, Kelly Rees, Ghinwa H Darwish, Jasmine Bernal-Escalante, W Russ Algar.

  The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological roles─substrate, receptor, and inhibitor─and were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.

Keywords:  Förster resonance energy transfer (FRET); biosensing; exosite; peptide; protease; quantum dot

DOI:  https://doi.org/10.1021/acsnano.4c03265
Int J Surg Case Rep. 2024 May 23. pii: S2210-2612(24)00570-4. [Epub ahead of print]120 109789

Metastatic melanoma to the urinary bladder with synchronous or metachronous metastasis to other organs: Clinicopathological characteristics and outcome.

Tu Anh Do, Nhung Thi Mai, Uyen Thi Le, Duong Ngoc Nguyen, Phuong Thanh Vu, Chu Van Nguyen.

   INTRODUCTION AND IMPORTANCE: Bladder metastatic melanoma is a very uncommon condition.
CASE PRESENTATION: On 62 reported cases, 55 studies have been done so far. We describe a 53-year-old woman with a hematuria who underwent transurethral resection of bladder lesions caused by metastatic melanoma for eight years ago after receiving her initial diagnosis of cutaneous malignant melanoma.
CLINICAL DISCUSSION: We also review the medical literature to determine the prognosis of bladder metastatic melanoma. Synchronous metastases with metastatic melanoma to the bladder also reduces the mean survival compared with patients with metachronous metastases.
CONCLUSION: Bladder metastatic melanoma combined with other factors, such as male, lymph node metastases, primary skin tumor, two or more bladder metastatic foci, and synchronous metastases are predictors of worse prognosis.

Keywords:  Bladder metastatic melanoma; Melanoma; Synchronous or Metachronous metastases

DOI:  https://doi.org/10.1016/j.ijscr.2024.109789