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The Journal of Biological Chemistry Dec 2019Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes... (Review)
Review
Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely ( during pathogen killing or enzymatic reactions) or accidentally ( exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.
Topics: Amino Acids; Animals; Anions; Antioxidants; Free Radicals; Humans; Kinetics; Nitric Oxide; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Peptides; Protein Processing, Post-Translational; Proteins; Reactive Oxygen Species; Ultraviolet Rays
PubMed: 31672919
DOI: 10.1074/jbc.REV119.006217 -
Endocrinology Feb 2022Hypothalamic kisspeptin (Kiss1) neurons provide indispensable excitatory transmission to gonadotropin-releasing hormone (GnRH) neurons for the coordinated release of... (Review)
Review
Hypothalamic kisspeptin (Kiss1) neurons provide indispensable excitatory transmission to gonadotropin-releasing hormone (GnRH) neurons for the coordinated release of gonadotropins, estrous cyclicity, and ovulation. But maintaining reproductive functions is metabolically demanding so there must be a coordination with multiple homeostatic functions, and it is apparent that Kiss1 neurons play that role. There are 2 distinct populations of hypothalamic Kiss1 neurons, namely arcuate nucleus (Kiss1ARH) neurons and anteroventral periventricular and periventricular nucleus (Kiss1AVPV/PeN) neurons in rodents, both of which excite GnRH neurons via kisspeptin release but are differentially regulated by ovarian steroids. Estradiol (E2) increases the expression of kisspeptin in Kiss1AVPV/PeN neurons but decreases its expression in Kiss1ARH neurons. Also, Kiss1ARH neurons coexpress glutamate and Kiss1AVPV/PeN neurons coexpress gamma aminobutyric acid (GABA), both of which are upregulated by E2 in females. Also, Kiss1ARH neurons express critical metabolic hormone receptors, and these neurons are excited by insulin and leptin during the fed state. Moreover, Kiss1ARH neurons project to and excite the anorexigenic proopiomelanocortin neurons but inhibit the orexigenic neuropeptide Y/Agouti-related peptide neurons, highlighting their role in regulating feeding behavior. Kiss1ARH and Kiss1AVPV/PeN neurons also project to the preautonomic paraventricular nucleus (satiety) neurons and the dorsomedial nucleus (energy expenditure) neurons to differentially regulate their function via glutamate and GABA release, respectively. Therefore, this review will address not only how Kiss1 neurons govern GnRH release, but how they control other homeostatic functions through their peptidergic, glutamatergic and GABAergic synaptic connections, providing further evidence that Kiss1 neurons are the key neurons coordinating energy states with reproduction.
Topics: Animals; Body Temperature Regulation; Brain Chemistry; Energy Metabolism; Female; Gonadotropin-Releasing Hormone; Homeostasis; Humans; Hypothalamus; Kisspeptins; Luteinizing Hormone; Neurons; RNA, Messenger; Reproduction
PubMed: 34953135
DOI: 10.1210/endocr/bqab253 -
The Journal of Biological Chemistry Oct 2023Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR...
Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAIN subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.
Topics: Cell Adhesion; Peptides; Protein Binding; Protein Domains; Receptors, G-Protein-Coupled; Signal Transduction; Humans
PubMed: 37673336
DOI: 10.1016/j.jbc.2023.105223 -
Chemistry (Weinheim An Der Bergstrasse,... Jan 2021Photochemical reactions have been the subject of renewed interest over the last two decades, leading to the development of many new, diverse and powerful chemical... (Review)
Review
Photochemical reactions have been the subject of renewed interest over the last two decades, leading to the development of many new, diverse and powerful chemical transformations. More recently, these developments have been expanded to enable the photochemical macrocyclisation of peptides and small proteins. These constructs benefit from increased stability, structural rigidity and biological potency over their linear counterparts, providing opportunities for improved therapeutic agents. In this review, an overview of both the established and emerging methods for photochemical peptide macrocyclisation is presented, highlighting both the limitations and opportunities for further innovation in the field.
Topics: Cyclization; Peptides; Photochemical Processes
PubMed: 32914455
DOI: 10.1002/chem.202003779 -
Toxins Feb 2021Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell... (Review)
Review
Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).
Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cell-Penetrating Peptides; Drug Carriers; Humans; Venoms
PubMed: 33671927
DOI: 10.3390/toxins13020147 -
Journal of the American Chemical Society Mar 2024There have been significant advances in the flexibility and power of cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and...
There have been significant advances in the flexibility and power of cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.
Topics: Biological Products; Cheminformatics; Peptides; Peptide Biosynthesis; Amino Acids
PubMed: 38470819
DOI: 10.1021/jacs.3c11306 -
Molecules (Basel, Switzerland) May 2023Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties....
Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the β-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation.
Topics: Molecular Dynamics Simulation; Amyloid; Molecular Conformation; Dimerization; Amyloid beta-Peptides; Peptide Fragments
PubMed: 37241811
DOI: 10.3390/molecules28104070 -
Nature Chemical Biology May 2024Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as...
Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics. In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability. In a proof of concept, we synthesized a library of 8,448 cyclic peptides and screened them against the disease target thrombin. Our workflow allowed multiple iterative cycles of library synthesis and yielded cyclic peptides with nanomolar affinities, high stabilities and an oral bioavailability (%F) as high as 18% in rats. This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics.
Topics: Peptides, Cyclic; Administration, Oral; Biological Availability; Animals; Rats; Humans; Cyclization; Peptide Library; Thrombin; Male; Combinatorial Chemistry Techniques; Acylation
PubMed: 38155304
DOI: 10.1038/s41589-023-01496-y -
International Journal of Molecular... Sep 2023Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association...
Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association and protein-protein interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered domain (STIL) and a structured CCD (STIL CCD). Disrupting STIL oligomerization via the CCD inhibits its activity We describe a comprehensive biophysical and structural characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (K = 8 ± 2 µM) and tetramerization (K = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order oligomers result in increased thermal stability and cooperativity of association. We suggest that this complex oligomerization mechanism regulates the activated levels of STIL in the cell and during centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.
Topics: Biophysical Phenomena; Circular Dichroism; Dimerization; Peptides; Protein Domains; Proteins; Humans; Intracellular Signaling Peptides and Proteins
PubMed: 37834064
DOI: 10.3390/ijms241914616 -
The Journal of Biological Chemistry Jun 2022Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the...
Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNA or Val-tRNA reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.
Topics: Peptide Biosynthesis; Peptides; Polylysine; RNA, Messenger; RNA, Transfer; RNA, Transfer, Lys; Ribosomes
PubMed: 35595100
DOI: 10.1016/j.jbc.2022.102039