Comparing Multifunctional Viral and Eukaryotic Proteins for Generating Scission Necks in Membranes.

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is M2 viroporin, a proton pump from the influenza A virus that is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging small-angle X-ray scattering structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemifission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Increased LL37 in psoriasis and other inflammatory disorders promotes LDL uptake and atherosclerosis.

Patients with chronic inflammatory disorders such as psoriasis have an increased risk of cardiovascular disease and elevated levels of LL37, a cathelicidin host defense peptide that has both antimicrobial and proinflammatory properties. To explore whether LL37 could contribute to the risk of heart disease, we examined its effects on lipoprotein metabolism and show that LL37 enhanced LDL uptake in macrophages through the LDL receptor (LDLR), scavenger receptor class B member 1 (SR-B1), and CD36. This interaction led to increased cytosolic cholesterol in macrophages and changes in expression of lipid metabolism genes consistent with increased cholesterol uptake. Structure-function analysis and synchrotron small-angle x-ray scattering showed structural determinants of the LL37-LDL complex that underlie its ability to bind its receptors and promote uptake. This function of LDL uptake is unique to cathelicidins from humans and some primates and was not observed with cathelicidins from mice or rabbits. Notably, Apoe-/- mice expressing LL37 developed larger atheroma plaques than did control mice, and a positive correlation between plasma LL37 and oxidized phospholipid on apolipoprotein B (OxPL-apoB) levels was observed in individuals with cardiovascular disease. These findings provide evidence that LDL uptake can be increased via interaction with LL37 and may explain the increased risk of cardiovascular disease associated with chronic inflammatory disorders.

Comparing multifunctional viral and eukaryotic proteins for generating scission necks in membranes.

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is the M2 viroporin, a proton pump from the influenza A virus which is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging Small-Angle X-ray Scattering (SAXS) structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemi-fission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

Apolipoprotein Mimetic Peptide Inhibits Neutrophil-Driven Inflammatory Damage via Membrane Remodeling and Suppression of Cell Lysis.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP1. X-ray structural studies and theoretical calculations show that apoMP1 induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and "turn off" diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP1 dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP1 inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter.

Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones-N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk-were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and ßIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, ßIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.

Sequence determinants in the cathelicidin LL-37 that promote inflammation via presentation of RNA to scavenger receptors.

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure-function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term "innate immune vetting" to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.

Tobacco smoking and risk of recurrence for squamous cell cancer of the anus.

OBJECTIVE: Squamous cell cancer of the anus is associated with multiple risk factors, including infection with human papillomavirus, immunosuppression, chronic inflammation, and tobacco smoking, although there is little data on these factors for the prediction of recurrent disease. Here, we evaluated the risk of recurrence and mortality of anal carcinoma in association with tobacco smoking. METHODS: We conducted a retrospective review of cases of anal carcinoma from two local hospitals. We obtained information on treatment response and cancer recurrence, as well as tobacco usage from medical records. RESULTS: We identified 64 patients with squamous cell cancer of the anus, and 34 of these (53%) had a tobacco smoking history. Current smokers had higher carcinoma recurrence rates (11/34, 32%) than non-smokers (6/30, 20%). Overall mortality was 33% (21/64), and cancer-related mortality was 23% (15/64). Smokers were more likely to die from recurrence than non-smokers, with 45% of smokers dead compared to only 20% of non-smokers by 5 years after treatment. CONCLUSION: Tobacco smoking appears to be associated with anal carcinoma disease recurrence, and is related to increased mortality. This data suggests that patients should be cautioned about tobacco smoking once a diagnosis of anal carcinoma is made in attempt to improve their long-term outcome.

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery.

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.