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Precision genetic medicine enlists antisense oligonucleotides (ASOs) to bind to nucleic acid targets important for human disease. Peptide nucleic acids (PNAs) have many desirable attributes as ASOs but lack cellular permeability. Here, we use an assay based on the corrective splicing of an mRNA to assess the ability of synthetic peptides to deliver a functional PNA into a human cell. We find that the endosomolytic peptides L17E and L17ER 4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER 4 enables robust corrective splicing in nearly all treated cells. Peptide-PNA conjugates are even more effective. These results enhance the utility of PNAs as research tools and potential therapeutic agents.
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Pentametaphosphate is the little studied cyclic pentamer of the metaphosphate ion, [PO3]5 5-. We show that the doubly protonated form of this pentamer can be selectively dehydrated to provide the anhydride [P5O14]3- (1). This trianion is the well-defined condensed phosphate component of a novel reagent for attachment of a pentaphosphate chain to biomolecules all in one go. Here, we demonstrate by extending adenosine monophosphate (AMP) and uridine monophosphate (UMP) to their corresponding nucleoside hexaphosphates, while adenosine diphosphate (ADP) and uridine diphosphate (UDP) are phosphate chain-extended to the corresponding nucleoside heptaphosphates. Such constructs are of interest for their potential biological function with respect to RNA-processing enzymes. Thus, we go on to investigate in detail the interaction of the polyanionic constructs with ribonuclease A, a model protein containing a polycationic active site and for which X-ray crystal structures are relatively straightforward to obtain. This work presents a combined experimental and quantum chemical approach to understanding the interactions of RNase A with the new nucleoside hexa- and heptaphosphate constructs.
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Approaches that leverage orthogonal chemical reactions to generate protein-protein conjugates have expanded access to bespoke chimeras. Although the literature is replete with examples of the semisynthesis of bispecific proteins, few methods exist for the semisynthesis of protein conjugates of higher complexity (i.e., greater than two-protein fusions). The recent emergence of trispecific cell engagers for immune cell redirection therapies necessitates the development of chemical methods for the construction of trispecific proteins that would otherwise be inaccessible via natural protein synthesis. Here, we demonstrate that 3-bromo-5-methylene pyrrolone (3Br-5MP) can be used to effect the facile chemical synthesis of trispecific peptides and proteins with exquisite control over the addition of each monomer. The multimeric complexes maintain epitope functionality both in human cells and upon immobilization. We anticipate that facile access to trispecific proteins using this 3Br-5MP will have broad utility in basic science research and will quicken the pace of research to establish novel, multimeric immune cell redirection therapies.
Assuntos
Proteínas , Humanos , Proteínas/química , Peptídeos/químicaRESUMO
Post-translational modifications (PTMs) increase the diversity of the proteome and are vital to organismal life and therapeutic strategies. Deep learning has been used to predict PTM locations. Still, limitations in datasets and their analyses compromise success. Here we evaluate the use of known PTM sites in prediction via sequence-based deep learning algorithms. Specifically, PTM locations were encoded as a separate amino acid before sequences were encoded via word embedding and passed into a convolutional neural network that predicts the probability of a modification at a given site. Without labeling known PTMs, our model is on par with others. With labeling, however, we improved significantly upon extant models. Moreover, knowing PTM locations can increase the predictability of a different PTM. Our findings highlight the importance of PTMs for the installation of additional PTMs. We anticipate that including known PTM locations will enhance the performance of other proteomic machine learning algorithms.
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Genetic studies indicate that breast cancer can be divided into several basic molecular groups. One of these groups, termed IntClust-2, is characterized by amplification of a small portion of chromosome 11 and has a median survival of only five years. Several cancer-relevant genes occupy this portion of chromosome 11, and it is thought that overexpression of a combination of driver genes in this region is responsible for the poor outcome of women in this group. In this study we used a gene editing method to knock out, one by one, each of 198 genes that are located within the amplified region of chromosome 11 and determined how much each of these genes contributed to the survival of breast cancer cells. In addition to well-known drivers such as CCND1 and PAK1 , we identified two different genes ( SERPINH1 and P4HA3 ), that encode proteins involved in collagen synthesis and organization. Using both in vitro and in vivo functional analyses, we determined that P4HA3 and/or SERPINH1 provide a critical driver function on IntClust-2 basic processes, such as viability, proliferation, and migration. Inhibiting these enzymes via genetic or pharmacologic means reduced collagen synthesis and impeded oncogenic signaling transduction in cell culture models, and a small-molecule inhibitor of P4HA3 was effective in treating 11q13 tumor growth in an animal model. As collagen has a well-known association with tissue stiffness and aggressive forms of breast cancer, we believe that the two genes we identified provide an opportunity for a new therapeutic strategy in IntClust-2 breast cancers.
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Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CLpro . Zymogen activation by 3CLpro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications.
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COVID-19 , Proteases 3C de Coronavírus , Vacinas , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Precursores Enzimáticos/genética , Ribonucleases , Pandemias , Proteínas não Estruturais Virais/química , Inibidores de Proteases/química , Antivirais/químicaRESUMO
The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.
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Peptídeos Penetradores de Células , Corantes Fluorescentes , Hidrolases , Humanos , Transporte Biológico , Peptídeos Penetradores de Células/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Corantes Fluorescentes/químicaRESUMO
"Click organocatalysis" uses mutually orthogonal click reactions to organocatalyze a click reaction. We report the development of an isobenzofuran organocatalyst that increases the rate and regioselectivity of an azide-alkyne cycloaddition. The organocatalytic cycle consists of (1) a Diels-Alder reaction of an alkyne with a diarylisobenzofuran to form a benzooxanorbornadiene, (2) a 1,3-dipolar cycloaddition with an azide to form a 4,5-dihydro-1,2,3-triazole, and (3) a retro-Diels-Alder reaction that releases the triazole product and regenerates the diarylisobenzofuran organocatalyst. The diarylisobenzofuran organocatalyst was computationally designed to catalyze the reaction of perfluorophenyl azide and methyl propiolate to selectively form a 1,4-triazole product. Experimental validation of the designed organocatalyst was obtained with methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl propiolate.