ABSTRACT
Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/metabolism , Escherichia coli/virology , Allosteric Regulation , Bacteriophage lambda/genetics , Bacteriophage lambda/physiology , CRISPR-Cas Systems , DNA Cleavage , DNA Restriction Enzymes/chemistry , Escherichia coli/enzymology , Escherichia coli/immunology , Genome, Viral , Protein Multimerization , Second Messenger SystemsABSTRACT
Synaptic dysfunction has been implicated as an early cause of cognitive decline in neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD). Methods to slow down or reverse the loss of functional synapses, therefore, represent a promising avenue to explore for treating NDDs. We have previously reported the development of a class of benzothiazole amphiphiles (BAMs) that exhibited the capability to improve memory and learning both in wild-type mice and in an AD rodent model, putatively through promoting RasGRF1-associated formation of dendritic spines in hippocampal neurons. While these results represent a good first step in exploring a new approach to treating NDDs, the capability of these compounds to increase spine density has not been previously examined in a human neuronal model. Here, we found that neurons derived from differentiated human induced pluripotent stem cells exhibited both an increase in RasGRF1 expression and a phenotypic increase in the density of postsynaptic density protein 95-positive puncta (which we use to provide an estimate of dendritic spine density) in BAM-treated vs. control neurons. These results demonstrate that the previously observed spinogenic effects of BAMs in rodent neurons can be recapitulated in a human neuronal model, which further supports the potential utility of BAM agents for treating human diseases associated with spine deficits such as AD or other NDDs.
Subject(s)
Benzothiazoles/pharmacology , Neurons/metabolism , ras-GRF1/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Benzothiazoles/metabolism , Brain/drug effects , Brain/metabolism , Brain/physiopathology , Cells, Cultured , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/physiopathology , Dendritic Spines/drug effects , Dendritic Spines/metabolism , Disks Large Homolog 4 Protein/analysis , Disks Large Homolog 4 Protein/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiopathology , Humans , Induced Pluripotent Stem Cells/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/physiopathology , Neurons/drug effects , Synapses/drug effects , Synapses/metabolism , ras-GRF1/drug effectsABSTRACT
Spider silk is a striking and robust natural material that has an unrivaled combination of strength and elasticity. There are two major problems in creating materials from recombinant spider silk proteins (rSSps): expressing sufficient quantities of the large, highly repetitive proteins and solvating the naturally self-assembling proteins once produced. To address the second problem, we have developed a method to rapidly dissolve rSSps in water in lieu of traditional organic solvents and accomplish nearly 100% solvation and recovery of the protein. Our method involves generating pressure and temperature in a sealed vial by using short, repetitive bursts from a conventional microwave. The method is scalable and has been successful with all rSSps used to date. From these easily generated aqueous solutions of rSSps, a wide variety of materials have been produced. Production of fibers, films, hydrogels, lyogels, sponges, and adhesives and studies of their mechanical and structural properties are reported. To our knowledge, ours is the only method that is cost-effective and scalable for mass production. This solvation method allows a choice of the physical form of product to take advantage of spider silks' mechanical properties without using costly and problematic organic solvents.
