Metabolic regulation of homologous recombination repair by MRE11 lactylation.

Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.

Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

HLA-B27.

Possession of the human leukocyte antigen (HLA) class I molecule B27 is strongly associated with ankylosing spondylitis (AS), but the pathogenic role of HLA-B27 is unknown. Two broad theories most likely explain the role of HLA-B27 in AS pathogenesis. The first is based on the natural immunological function of HLA-B27 of presenting antigenic peptides to cytotoxic T cells. Thus, HLA-B27-restricted immune responses to self-antigens, or arthritogenic peptides, might drive immunopathology. B27 can also "behave badly," misfolding during assembly and leading to endoplasmic reticulum stress and autophagy responses. ß2m-free B27 heavy chain structures including homodimers (B272) can also be expressed at the cell surface following endosomal recycling of cell surface heterotrimers. Cell surface free heavy chains and B272 bind to innate immune receptors on T, NK, and myeloid cells with proinflammatory effects. This review describes the natural function of HLA-B27, its disease associations, and the current theories as to its pathogenic role.

A male-derived nonribosomal peptide pheromone controls female schistosome development.

Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates ß-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic ß-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.

Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Extracellular pH sensing by plant cell-surface peptide-receptor complexes.

The extracellular pH is a vital regulator of various biological processes in plants. However, how plants perceive extracellular pH remains obscure. Here, we report that plant cell-surface peptide-receptor complexes can function as extracellular pH sensors. We found that pattern-triggered immunity (PTI) dramatically alkalinizes the acidic extracellular pH in root apical meristem (RAM) region, which is essential for root meristem growth factor 1 (RGF1)-mediated RAM growth. The extracellular alkalinization progressively inhibits the acidic-dependent interaction between RGF1 and its receptors (RGFRs) through the pH sensor sulfotyrosine. Conversely, extracellular alkalinization promotes the alkaline-dependent binding of plant elicitor peptides (Peps) to its receptors (PEPRs) through the pH sensor Glu/Asp, thereby promoting immunity. A domain swap between RGFR and PEPR switches the pH dependency of RAM growth. Thus, our results reveal a mechanism of extracellular pH sensing by plant peptide-receptor complexes and provide insights into the extracellular pH-mediated regulation of growth and immunity in the RAM.

State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides.

The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.

Emergence of immune escape at dominant SARS-CoV-2 killer T cell epitope.

We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as "variants of concern," was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants.

Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

An Excitatory Circuit in the Perioculomotor Midbrain for Non-REM Sleep Control.

The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.

Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors.

The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.

A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain.

TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.

T-Scan: A Genome-wide Method for the Systematic Discovery of T Cell Epitopes.

T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses.

Protein Sequence Editing of SKN-1A/Nrf1 by Peptide:N-Glycanase Controls Proteasome Gene Expression.

The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an endoplasmic reticulum (ER)-associated transcription factor that undergoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.

Reductionist Approach in Peptide-Based Nanotechnology.

The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like ß-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.