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Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax., Competing Interests: Declaration of interests T.O. received research funding from Gilead (related to this work) and Merck KGaA (not related to this work). T.O. is a consultant for Roche and Merck KGaA (both not related to this work). K.S. receives grant funding as part of the DFCI/Novartis Drug Discovery Program, consults for and has stock options in Auron Therapeutics, and has consulted for Kronos Bio and AstraZeneca on topics not directly related to this manuscript. F.C. is a co-founder of enGene Statistics GmbH. The Max Planck institute and the Goethe University Frankfurt are filing a patent application, on which T.O., A.K.J., S.Wolf, F.B., H.S., M.M., and H.U. are listed as inventors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495-2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium., (© 2021. The Author(s).)

Background: Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, [Formula: see text]. Previously reported experiments have suggested that 3-5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the [Formula: see text]. The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity., Results: In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans' strain pathogenicity or growth form., Conclusions: Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the [Formula: see text] might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity., (© 2021. The Author(s).)

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro , our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen., (© 2019 Chachami et al.)

Post-transcriptional gene regulation controls the amount of protein produced from a specific mRNA by altering both its decay and translation rates. Such regulation is primarily achieved by the interaction of trans-acting factors with cis-regulatory elements in the untranslated regions (UTRs) of mRNAs. These interactions are guided either by sequence- or structure-based recognition. Similar to sequence conservation, the evolutionary conservation of a UTR's structure thus reflects its functional importance. We used such structural conservation to identify previously unknown cis-regulatory elements. Using the RNA folding program Dynalign, we scanned all UTRs of humans and mice for conserved structures. Characterizing a subset of putative conserved structures revealed a binding site of the RNA-binding protein Roquin. Detailed functional characterization in vivo enabled us to redefine the binding preferences of Roquin and identify new target genes. Many of these new targets are unrelated to the established role of Roquin in inflammation and immune responses and thus highlight additional, unstudied cellular functions of this important repressor. Moreover, the expression of several Roquin targets is highly cell-type-specific. In consequence, these targets are difficult to detect using methods dependent on mRNA abundance, yet easily detectable with our unbiased strategy.

Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Although the "universal" genetic code is now known not to be universal, and stop codons can have multiple meanings, one regularity remains, namely that for a given sense codon there is a unique translation. Examining CUG usage in yeasts that have transferred CUG away from leucine, we here report the first example of dual coding: Ascoidea asiatica stochastically encodes CUG as both serine and leucine in approximately equal proportions. This is deleterious, as evidenced by CUG codons being rare, never at conserved serine or leucine residues, and predominantly in lowly expressed genes. Related yeasts solve the problem by loss of function of one of the two tRNAs. This dual coding is consistent with the tRNA-loss-driven codon reassignment hypothesis, and provides a unique example of a proteome that cannot be deterministically predicted. VIDEO ABSTRACT., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The genetic code is a set of instructions that determine how the information in our genetic material is translated into amino acids. In general, it is universal for all organisms, from viruses and bacteria to humans. However, in the last few decades, exceptions to this rule have been identified both in pro- and eukaryotes. In this review, we discuss the 16 described alternative eukaryotic nuclear genetic codes and observe theories of their appearance in evolution. We consider possible molecular mechanisms that allow codon reassignment. Most reassignments in nuclear genetic codes are observed for stop codons. Moreover, in several organisms, stop codons can simultaneously encode amino acids and serve as termination signals. In this case, the meaning of the codon is determined by the additional factors besides the triplets. A comprehensive review of various non-standard coding events in the nuclear genomes provides a new insight into the translation mechanism in eukaryotes., (© 2024 Wiley Periodicals LLC.)

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects., (© 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.)

The promyelocytic leukemia protein (PML) and its associated nuclear bodies have recently emerged as critical regulators of embryonic stem (ES) cell identity. Despite their recognized importance, the complete spectrum of PML-mediated molecular events in ES cells remains unclear. In this report, we study how PML is shaping the proteomic and SUMO proteomic landscape in ES cells. Proteomic profiling of PML-depleted ES cells uncovered a downregulation of self-renewal factors and an upregulation of proteins associated with translation and proteasomal activity, reflecting a cellular transition from pluripotency to differentiation. Importantly, PML promotes the sumoylation of pluripotency-related factors, chromatin organizers, and cell cycle regulators. We identified SALL1 and CDCA8 as novel PML-directed sumoylation targets, both critical for ES cell maintenance. SALL1 sumoylation increases the activation of the Wnt pathway, contributing to its ability to inhibit ES cell differentiation. Similarly, CDCA8 sumoylation enhances its capacity to promote cell proliferation. Collectively, our findings demonstrate that PML regulates ES cell identity by modulating the abundance or sumoylation of key regulators involved in pluripotency and cell cycle progression. [ABSTRACT FROM AUTHOR]

Rice plants are important food crops that are sensitive to cold stress. Microtubules (MTs) are highly associated with plant response to cold stress. The exogenous application of abscisic acid (ABA) can transiently induce the cold stability of microtubules. These phenotypes were accompanied by the transient increase in Phospholipase D (PLD) enzyme activity. The analysis of detyrosinated/tyrosinated α-tubulin by Western blot in the NtTUA3 line or in the NtTUA3+OsTTL line gave us such a conclusion that the effect of ABA on detyrosinated α-tubulin not only was regulated by ABA but also was dependent on the TTLL12 protein. The dual ABA and 1% n-butanol treatments had shown that ABA-induced detyrosinated α-tubulin in a manner distinct from the n-butanol pathway. Detecting the detyrosinated α-tubulin level after pre-treatment with pertussis toxin (PTX), a G-protein inhibitor, followed by ABA, as well as mastoparan (Mas7) treatment suggested that the effect of ABA on detyrosinated α-tubulin was dependent on PLD activity. [ABSTRACT FROM AUTHOR]

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue., ((c) 2010 by John Wiley & Sons, Inc.)

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.

UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA. In combination with modern mass spectrometric (MS) analysis such an approach provides the opportunity to reveal not only the nature of the crosslinked proteins but also to identify the actual crosslinking sites between the protein and the nucleic acid. However, the relatively low yield in UV-induced crosslinking makes it difficult to identify in particular those species by MS that represent peptide-nucleic acid conjugates, as the great excess of noncrosslinked material interferes with their detection in MS. Here, we present an automated enrichment strategy of crosslinked peptide-RNA oligonucleotides derived from crude mixtures of UV-irradiated ribonucleoprotein (RNP) particles that uses TiO(2) columns integrated within a two-dimensional (2D) nanoliquid chromatography (LC) system. The setup combines two C18 precolumns, a TiO(2) enrichment column and a nanoanalytical column. It allows the removal of the noncrosslinked RNA and protein moiety and the specific enrichment of crosslinked peptide-RNA conjugates so that UV-irradiated and subsequently completely hydrolyzed RNP complexes can directly be loaded and analyzed by MS. In this feasibility study, we demonstrate the specific enrichment of peptide-RNA oligonucleotides derived from UV-irradiated native spliceosomal U1 snRNPs and spliceosomal [15.5K-61K-U4atac snRNA] complex reconstituted in vitro., (2008 Wiley Periodicals, Inc)

A new atmospheric pressure (AP)-MALDI-type interface has been developed based on a free liquid (FL) microbeam/microdroplets and a mid-infrared optical parametric oscillator (mid-IR OPO). The device is integrated into a standard on-line nanoESI interface. The generation of molecular ions in the gas phase is believed to be the result of a fast (explosive) laser-induced evaporative dispersion(not desorption) of the microbeam into statistically charged nanodroplets. Only the lowest charge states appear insignificant abundance in this type of experiment. Mass spectra of some common peptides have been acquired in positive ion mode, and the limit-of-detection of this first prototype (liquid microbeam setup) was evaluated to be 17 fmol per second. To improve the duty cycle and to reduce the sample consumption, a droplet-on-demand system was implemented (generating 100 pL droplets).With this setup, about 20 attomole of bradykinin were sufficient to achieve a signal-to-noise ratio better than five.This setup can be operated at flow rates down to 100 nL/min and represents a liquid MALDI alternative to the nanoESI. Our particular interest was the application of the developed ion source for on-line coupling of liquid chromatography with mass spectrometry. The flow rates(>100 microL/min), required for stable operation of the ion source in continuous liquid microbeam mode, matches perfectly the flow rate range of micro HPLC. Therefore, online LC/MS experiments have been realized, employing a microbore C18 reversed-phase column to separate an artificial peptide mixture and tryptic peptides of bovine serum albumin (performing a peptide mass fingerprint). In the latter case, sequence coverage of more than 90%has been achieved.

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.

The translation of nucleotide sequences into amino acid sequences, governed by the genetic code, is one of the most conserved features of molecular biology. The standard genetic code, which uses 61 sense codons to encode one of the 20 standard amino acids and 3 stop codons (UAA, UAG, and UGA) to terminate translation, is used by most extant organisms. The protistan phylum Ciliophora (the 'ciliates') are the most prominent exception to this norm, exhibiting the grfeatest diversity of nuclear genetic code variants and evidence of repeated changes in the code. In this study, we report the discovery of multiple independent genetic code changes within the Phyllopharyngea class of ciliates. By mining publicly available ciliate genome datasets, we discovered that three ciliate species from the TARA Oceans eukaryotic metagenome dataset use the UAG codon to putatively encode leucine. We identified novel suppressor tRNA genes in two of these genomes which are predicted to decode the reassigned UAG codon to leucine. Phylogenomics analysis revealed that these three uncultivated taxa form a monophyletic lineage within the Phyllopharyngea class. Expanding our analysis by reassembling published phyllopharyngean genome datasets led to the discovery that the UAG codon had also been reassigned to putatively code for glutamine in Hartmannula sinica and Trochilia petrani. Phylogenomics analysis suggests that this occurred via two independent genetic code change events. These data demonstrate that the reassigned UAG codons have widespread usage as sense codons within the phyllopharyngean ciliates. Furthermore, we show that the function of UAA is firmly fixed as the preferred stop codon. These findings shed light on the evolvability of the genetic code in understudied microbial eukaryotes. Author summary: The genetic code dictates the translation of nucleotide sequences into amino acids. It is one of the most conserved features of molecular biology. Most organisms use the "standard genetic code", where 61 codons encode amino acids and three stop codons (UAA, UAG, and UGA) signal translation termination. Ciliates–microbial single-celled eukaryotes–are the most prominent exception, with multiple lineages exhibiting variant genetic codes in which one or more stop codons have been reassigned to function as sense codons. In this study, we report novel genetic code changes in poorly studied ciliates. By analysing marine metagenomic data from the TARA Oceans Project, we identified an uncultivated lineage of ciliates from the Phyllopharyngea class that uses the UAG codon to encode leucine. Extending our analysis to include other genomes from the Phyllopharyngea class, we identified further changes in Hartmannula sinica and Trochilia petrani where the UAG codon had been reassigned to encode glutamine. Phylogenomics analysis suggests that three lineages within Phyllopharyngea independently reassigned the codon UAG to encode an amino acid. These findings expand our understanding of genetic code evolution and highlight the remarkable diversity of genetic codes employed by ciliates. [ABSTRACT FROM AUTHOR]

Background: Developmental and epileptic encephalopathy (DEE) is a group of neurodevelopmental disorders characterized by early-onset seizures predominantly attributed to genetic causes. Nevertheless, numerous patients remain without identification of a genetic cause. Methods: We present four unrelated Chinese patients with SPOUT1 compound heterozygous variants, all of whom were diagnosed with DEE. We also investigated functions of SPOUT1 using the spout1 knockout zebrafish model. Results: The four unrelated DEE patients with SPOUT1 compound heterozygous variants were all males. Their onset age of seizure ranged from 3 months to 6 months (median age 5 months). All patients had epileptic spasms, and were diagnosed with infantile epileptic spasms syndrome (IESS). Three patients had microcephaly during infancy. Brain MRI in three patients showed white matter hypomyelination and bilaterally widened frontotemporal subarachnoid space. At the last follow-up, two patients exhibited drug-resistant epilepsy, one achieved seizure freedom following vigabatrin treatment, and one died at the age of 4 years and 5 months from probable sudden unexpected death in epilepsy. Seven different SPOUT1 variants were identified in the four patients, including six missense variants and one deletion variant. AlphaFold2 prediction indicated that all variants alternated the number or the length of bonds between animo acids in protein SPOUT1. Neurophysiological results from spout1 knockout zebrafish revealed the presence of epileptiform signals in 9 out of 63 spout1 knockout zebrafishes (P = 0.009). Transcriptome sequencing revealed 21 differentially expressed genes between spout1 knockout and control groups, including 13 up-regulated and 8 down-regulated genes. Two axonal transport-related genes, kif3a and ap3d1, were most prominently involved in enriched Gene Ontology (GO) terms. Conclusions: This study identified SPOUT1 as a novel candidate gene of DEE, which follows the autosomal-recessive inheritance pattern. IESS is the most common epilepsy syndrome. Downregulation of axonal transport-related genes, KIF3A and AP3D1, may play a crucial role in the pathogenesis of DEE. [ABSTRACT FROM AUTHOR]

MicroRNAs play a significant role in the development of cancers, including lung cancer. A recent study revealed that smoking, a key risk factor for lung cancer, increased the levels of hsa-mir-301a in the tumor tissues of patients with lung squamous cell carcinoma (LUSC). The aim of the current study is to investigate the mechanism by which tobacco smoke increases hsa-mir-301a levels in LUSC tumor tissues using bioinformatics analysis. Bioinformatics tools and online databases, including The Cancer Genome Atlas (TCGA), LinkedOmics, and Encyclopedia of RNA Interactomes (ENCORI), were applied in this study. Our results showed a correlation between the upregulation of hsa-mir-301a in LUSC tissues and smoking exposure. However, no correlation was discovered between patients' smoking status and the expression level of the hsa-mir-301a host gene, SKA2, prompting us to investigate possible changes in microRNA processing under tobacco smoke exposure. In silico results using online platforms suggest that post-transcriptional processes, which involve the RNA-binding proteins DGCR8 and FUS, contribute to the elevation of mature hsa-mir-301a levels in smoking patients with LUSC. Our findings suggest that RNA-binding proteins play a key role in controlling the processing of hsa-mir-301a, indicating a complex regulation of hsa-mir-301a in the LUSC tissues of smokers. [ABSTRACT FROM AUTHOR]

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNAs (miRNAs) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNAs, the roles of other miRNA-binding proteins (miRBPs) remain unclear in the regulation of miRNA loading, dissociation from RISCs and extracellular release. In this study, we performed protein arrays to profile miRBPs and identify 118 RBPsthat directly bind to miRNAs. Among those proteins, the RBP quaking (QKI) inhibits extracellular release of the mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as AUF1 (also known as hnRNPD) and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates Toll-like receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in the mouse cortex. Thus, this study reveals the contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release. [ABSTRACT FROM AUTHOR]

Bone health is ensured by the coordinated action of two types of cells—the osteoblasts that build up bone structure and the osteoclasts that resorb the bone. The loss of balance in their action results in pathological conditions such as osteoporosis. Central to this study is a class of RNA-binding proteins (RBPs) that regulates the biogenesis of miRNAs. In turn, miRNAs represent a critical level of regulation of gene expression and thus control multiple cellular and biological processes. The impact of miRNAs on the pathobiology of various multifactorial diseases, including osteoporosis, has been demonstrated. However, the role of RBPs in bone remodeling is yet to be elucidated. The aim of this study is to dissect the transcriptional landscape of genes encoding the compendium of 180 RBPs in bone cells. We developed and applied a multi-modular integrative analysis algorithm. The core methodology is gene expression analysis using the GENEVESTIGATOR platform, which is a database and analysis tool for manually curated and publicly available transcriptomic data sets, and gene network reconstruction using the Ingenuity Pathway Analysis platform. In this work, comparative insights into gene expression patterns of RBPs in osteoblasts and osteoclasts were obtained, resulting in the identification of 24 differentially expressed genes. Furthermore, the regulation patterns upon different treatment conditions revealed 20 genes as being significantly up- or down-regulated. Next, novel gene–gene associations were dissected and gene networks were reconstructed. Additively, a set of osteoblast- and osteoclast-specific gene signatures were identified. The consolidation of data and information gained from each individual analytical module allowed nominating novel promising candidate genes encoding RBPs in osteoblasts and osteoclasts and will significantly enhance the understanding of potential regulatory mechanisms directing intracellular processes in the course of (patho)physiological bone turnover. [ABSTRACT FROM AUTHOR]

The posttranslational modifications of alpha-tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. Alpha-Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii alpha-tubulin. The data suggest that the site of glutamylation on alpha-tubulin is conserved over a broad range of species.

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.

The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.