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There are errors in the third paragraph under the subheading \"Molecular dynamics simulations and analysis\" in the Methods section. In order to determine the torsion angle ϕ between two filament axes, the vectors between the Cα atoms of Phe19 in the two outmost filament layers were computed, the respective torsion angle between the ith and jth filament was calculated and averaged over all three filament pairs: 1.

Misuse and overuse of antibiotics have contributed in the last decades to a phenomenon known as antibiotic resistance which is currently considered one of the principal threats to global public health by the World Health Organization. The aim to find alternative drugs has been demonstrated as a real challenge. Thanks to their biodiversity, insects represent the largest class of organisms in the animal kingdom. The humoral immune response includes the production of antimicrobial peptides (AMPs) that are released into the insect hemolymph after microbial infection. In this review, we have focused on insect immune responses, particularly on AMP characteristics, their mechanism of action and applications, especially in the biomedical field. Furthermore, we discuss the Toll, Imd, and JAK-STAT pathways that activate genes encoding for the expression of AMPs. Moreover, we focused on strategies to improve insect peptides stability against proteolytic susceptibility such as D-amino acid substitutions, N-terminus modification, cyclization and dimerization.

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes. Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of inflammatory diseases. Here authors report a cryoelectron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, which provides insights into PACAP27 binding and VIP receptor activation.

A variety of chemical and enzymatic techniques, each with their own considerations for use, have been developed for the site-selective bioconjugation of desirable moieties to proteins via the unique handle of the N terminus. The formation of well-defined protein bioconjugates is critical for many studies and technologies in chemical biology. Tried-and-true methods for accomplishing this typically involve the targeting of cysteine residues, but the rapid growth of contemporary bioconjugate applications has required an expanded repertoire of modification techniques. One very powerful set of strategies involves the modification of proteins at their N termini, as these positions are typically solvent exposed and provide chemically distinct sites for many protein targets. Several chemical techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. A growing number of N-terminus-specific enzymatic ligation strategies have provided additional possibilities. This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each. Examples of specific N-terminal protein conjugates are provided, along with their uses in a number of diverse biological applications.

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T  = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids. Adeno-associated viruses (AAVs) have emerged as promising gene therapy vectors.The AAV capsid consists of 60 subunits made up from three distinct viral proteins (VPs). Here authors record high-resolution native mass spectra of intact AAV capsids to assess the VP stoichiometries in a panel of serotypes and reveals an extremely heterogeneous population of capsids of variable composition.

Protein ubiquitination has become one of the most extensively studied post-translational modifications. Originally discovered as a critical element in highly regulated proteolysis, ubiquitination is now regarded as essential for many other cellular processes. This results from the unique features of ubiquitin (Ub) and its ability to form various homo- and heterotypic linkage types involving one of the seven different lysine residues or the free amino group located at its N-terminus. While K48- and K63-linked chains are broadly covered in the literature, the other types of chains assembled through K6, K11, K27, K29, and K33 residues deserve equal attention in the light of the latest discoveries. Here, we provide a concise summary of recent advances in the field of these poorly understood Ub linkages and their possible roles in vivo.

Summary Plant‐specific NAC proteins constitute a major transcription factor family that is well‐known for its roles in plant growth, development, and responses to abiotic and biotic stresses. In recent years, there has been significant progress in understanding the functions of NAC proteins. NAC proteins have a highly conserved DNA‐binding domain; however, their functions are diverse. Previous understanding of the structure of NAC transcription factors can be used as the basis for their functional diversity. NAC transcription factors consist of a target‐binding domain at the N‐terminus and a highly versatile C‐terminal domain that interacts with other proteins. A growing body of research on NAC transcription factors helps us comprehend the intricate signalling network and transcriptional reprogramming facilitated by NAC‐mediated complexes. However, most studies of NAC proteins have been limited to a single function. Here, we discuss the upstream regulators, regulatory components and targets of NAC in the context of their prospective roles in plant improvement strategies via biotechnology intervention, highlighting the importance of the NAC transcription factor family in plants and the need for further research.

Synergistically improving T-cell responsiveness is promising for favorable therapeutic outcomes in immunologically cold tumors, yet current treatments often fail to induce a cascade of cancer-immunity cycle for effective antitumor immunity. Gasdermin-mediated pyroptosis is a newly discovered mechanism in cancer immunotherapy; however, cleavage in the N terminus is required to activate pyroptosis. Here, we report a single-agent mRNA nanomedicine-based strategy that utilizes mRNA lipid nanoparticles (LNPs) encoding only the N-terminus of gasdermin to trigger pyroptosis, eliciting robust antitumor immunity. In multiple female mouse models, we show that pyroptosis-triggering mRNA/LNPs turn cold tumors into hot ones and create a positive feedback loop to promote antitumor immunity. Additionally, mRNA/LNP-induced pyroptosis sensitizes tumors to anti-PD-1 immunotherapy, facilitating tumor growth inhibition. Antitumor activity extends beyond the treated lesions and suppresses the growth of distant tumors. We implement a strategy for inducing potent antitumor immunity, enhancing immunotherapy responses in immunologically cold tumors. mRNA nanomedicine-based gene therapy may offer opportunities for cancer treatment. Here the authors show that mRNA lipid nanoparticles encoding the N-terminal domain of gasdermin B trigger pyroptosis and promote anti-tumor immune responses in preclinical cancer models.

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1–121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50–57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1–Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson’s disease.