How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is ...complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.
•247 VOCs in Ganoderma were identified by HS-SPME-GC×GC-TOFMS for first.•The VOCs of Ganoderma obtained from four drying methods had a significant difference.•A number of specific VOCs in Ganoderma ...lucidum were excavated.
The effect of four drying methods (namely, sun drying, vacuum freeze drying, vacuum drying, and hot air drying) on the fresh Ganoderma lucidum volatile organic compounds (VOCs) was studied by their separation via headspace solid-phase microextraction and identification via comprehensive two-dimensional chromatography-time-of-flight mass spectrometry. In total, 247 kinds of VOCs, mainly composed of alcohols, aldehydes, esters, ketones, and olefins, were identified. Orthogonal partial least squares discriminant analysis and displacement test combined with the variable important in the projection method revealed that VOCs' types and contents in Ganoderma lucidum were influenced by the particular drying method, which could be identified using specific organic compounds. The heating process increased the content of aldehydes, esters, and olefins and reduced alcohol and ketones' content. Since 3-propoxy-1-Propene, 2-pentyl-Furan, Acetic acid, 2-Butanone, 1-Pantano, and other VOCs did not disappear during the drying process, they can be used for the classification and identification of Ganoderma lucidum species and other edible fungi.
MITA is a central adaptor in innate immune responses to DNA viruses. The mechanisms responsible for recruitment of downstream kinase TBK1 and the transcription factor IRF3 to MITA remains enigmatic. ...Here we identified ZDHHC11, a member of DHHC palmitoyl transferase family, as a positive regulator of DNA virus-triggered signaling. Overexpression of ZDHHC11 activated the IFN-β promoter, while ZDHHC11-deficiency specifically impaired DNA virus HSV-1-induced transcription of downstream antiviral genes. Zdhhc11
mice exhibited lower serum cytokine levels and higher lethality after HSV-1 infection. Mechanistically, ZDHHC11 facilitated the optimal recruitment of IRF3 to MITA. Our findings support an important role for ZDHHC11 in mediating MITA-dependent innate immune responses against DNA viruses.
The fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a ...novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage.
As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism ...has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNA
), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNA
depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNA
-specific 2'-
-methylation at the wobble position, making virtually all eukaryotic tRNA
susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.
The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA ...translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis.
Balanced protein synthesis and degradation are crucial for proper cellular function. Protein synthesis is tightly coupled to energy status and nutrient levels by the mammalian target of rapamycin ...complex 1 (mTORC1). Quality of newly synthesized polypeptides is maintained by the molecular chaperone and ubiquitin-proteasome systems. Little is known about how cells integrate information about the quantity and quality of translational products simultaneously. We demonstrate that cells distinguish moderate reductions in protein quality from severe protein misfolding using molecular chaperones to differentially regulate mTORC1 signaling. Moderate reduction of chaperone availability enhances mTORC1 signaling, whereas stress-induced complete depletion of chaperoning capacity suppresses mTORC1 signaling. Molecular chaperones regulate mTORC1 assembly in coordination with nutrient availability. This mechanism enables mTORC1 to rapidly detect and respond to environmental cues while also sensing intracellular protein misfolding. The tight linkage between protein quality and quantity control provides a plausible mechanism coupling protein misfolding with metabolic dyshomeostasis.
Abstract
Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the ...serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored ‘dark proteome’ is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.
In this issue, Diamond et al.1 and Kim et al.2 report that depletion of eIF4E leads to translational upregulation of GCN4, a key player in the integrated stress response, in an eIF2α ...phosphorylation-independent manner, suggesting a new mode of translational adaptation.
In this issue, Diamond et al. and Kim et al. report that depletion of eIF4E leads to translational upregulation of GCN4, a key player in the integrated stress response, in an eIF2α phosphorylation-independent manner, suggesting a new mode of translational adaptation.
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