Friedmandavid5878

Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induces IL23A expression in intestinal epithelial cells. Moreover, we identified a strong cross-talk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a non-canonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely becauseIL12B expression is absent in CRC cells. Given recent evidence that IL23Apromotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600Emutations. PGE2 purchase Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a non-canonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy (AFM) to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our AFM analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli.  We posit that the VEGFR2-AMPK-PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.There are a number of riboswitches that utilize the same ligand-binding domain to regulate either transcription or translation. S-box (SAM-I) riboswitches, including the riboswitch present in the Bacillus subtilis metI gene, which encodes cystathionine γ-synthase, regulate the expression of genes involved in methionine metabolism in response to SAM, primarily at the level of transcriptional attenuation. A rarer class of S-box riboswitches is predicted to regulate translation initiation. Here, we identified and characterized a translational S-box riboswitch in the metI gene from Desulfurispirillum indicum The regulatory mechanisms of riboswitches are influenced by the kinetics of ligand interaction. The half-life of the translational D. indicum metI RNA-SAM complex is significantly shorter than that of the transcriptional B. subtilis metI RNA. This finding suggests that unlike the transcriptional RNA, the translational metI riboswitch can make multiple reversible regulatory decisions. Comparison of both RNAs revealed that the second internal loop of helix P3 in the transcriptional RNA usually contains an A residue, whereas the translational RNA contains a C residue that is conserved in other S-box RNAs that are predicted to regulate translation. Mutational analysis indicated that the presence of an A or C residue correlates with RNA-SAM complex stability. These analyses indicate that the internal loop sequence critically determines the stability of the RNA-SAM complex by influencing the flexibility of residues involved in SAM binding and thereby affects the molecular mechanism of riboswitch function. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays.  Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated.  Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component.  Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b.  Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin.  We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components, and that CALML4 can also associate with the Usher complex component myosin-7a.  Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex, and may also shed light on the etiology of the sensory disorder USH1H. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Assembled a-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of a-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of a-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T a-synuclein (line M83) that seed aggregation. The most potent fractions contained sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD and six cases of multiple system atrophy (MSA) for their ability to induce a-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain sarkosyl-insoluble a-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled a-synuclein in these different samples. We conclude that a-synuclein are the main driving force for amplification and propagation of pathology in synucleinopathies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.OBJECTIVE To evaluate the efficacy and safety of eptinezumab, a humanized anti-calcitonin gene-related peptide monoclonal antibody, in the preventive treatment of chronic migraine (CM). METHODS The Prevention of Migraine via Intravenous ALD403 Safety and Efficacy-2 (PROMISE-2) study was a phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Adults with CM were randomly assigned to receive IV eptinezumab 100 mg, eptinezumab 300 mg, or placebo administered on day 0 and week 12. The primary endpoint was change from baseline in mean monthly migraine days (MMDs) over weeks 1 to 12. RESULTS Among treated participants (n = 1,072), baseline mean number of MMDs was ≈16.1 across groups. Treatment with eptinezumab 100 and 300 mg was associated with significant reductions in MMDs across weeks 1 to 12 compared with placebo (placebo -5.6, 100 mg -7.7, p 2% over placebo; it occurred in the 300 mg eptinezumab arm (eptinezumab 9.4%, placebo 6.0%). CONCLUSION In patients with CM, eptinezumab 100 and 300 mg was associated with a significant reduction in MMDs from the day after IV administration through week 12, was well tolerated, and demonstrated an acceptable safety profile.