Signature Motif Research Articles

Abstract PR020: A novel framework for epigenome-dependent multimodal fragment-signature analysis reveals insights into cell-free DNA generation in cancer

Abstract Background: The genome-wide pattern of cell-free DNA (cfDNA) is influenced by fragmentation enzymes, nucleosome occupancy, and epigenetic factors, as well as cell death mechanisms. Better modeling of these factors and their interactions will be essential for more sensitive detection of tumor-specific features and tissue-of-origin. Methods: To obtain mechanistic insights into cfDNA fragmentation, we developed a fragmentomics signature analysis framework. Our fragmenTopics algorithm (i) decomposes contributions of different fragment lengths and end-motif signatures using topic modeling and (ii) learns the dependencies of signature rates on the epigenome. The second component utilizes gradient boosting, enabling the modeling of non-linear dependencies and feature interactions. Both large-scale features, such as histone marks and DNase activity, and meso-scale features, such as transcription start sites (TSSs), nucleosome positions, and secondary DNA structures, are included in the model. Results: Using de novo signature discovery from 726 samples across five datasets, we built a comprehensive catalog of fragment length and end-motif signatures and studied determinants of their activity across the genome. The most common length and end-motif signatures reflected the mononucleosomal fragmentation by DNASE1L3. These signatures, as well as the dinucleotide length signature and motif signatures associated with known motifs of DNase enzymes, such as DFFB, showed lower abundance in cancer and further decreased with increasing tumor fraction. Instead, cancer samples manifested a higher diversity of processes, many of which are of unknown origin. Notably, two cancer-enriched length signatures were, on average, 20 bp shorter fragments with respect to mono- and di-nucleosome peaks. These signatures correlated with H3K4me1 and the compartment eigenvector, and may be linked to tighter nucleosome wrapping in euchromatin. Two cancer-enriched signatures (190-240 bp and 240-300 bp) had higher rates in TSSs; in addition to the two tight mono- and di-nucleosomal signatures, which were also enriched. Cancer-enriched end-motifs were A/T-rich, and these did not correspond to known DNase motifs. The increased diversity in end-motifs may be due to non-apoptotic cell death or tissue- or cancer-specific activity of less-studied cleavage enzymes; our results on their epigenome correlations can inform further investigations into their etiology. Conclusion: Our signature-based fragmentomics framework, which decouples the contributions of distinct processes while identifying the epigenomic determinants of their genome-wide rates, can improve our understanding of cell-free DNA fragmentation in cancer. Citation Format: Yoo-Na Kim, Sangmi Lee, Allen Lynch, Tae Hee Kim, Peter J Park, Doga Gulhan. A novel framework for epigenome-dependent multimodal fragment-signature analysis reveals insights into cell-free DNA generation in cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR020.

Characterizing Host microRNA: Virus Interactions of Orthoavulavirus javaense

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) relies on sequence complementarity between the miRNA seed site and the target gene transcript(s). This complementarity can completely inhibit or reduce translation into protein. We hypothesized that viruses employ sequence complementarity/similarity with host miRNAs to inhibit or increase the miRNA-mediated regulation of host gene expression specifically during viral infection(s). In this study, we focus on Orthoavulavirus javaense (OAVJ), the causative of Newcastle disease, a poultry disease with significant economic impact. A computational analysis of OAVJ genomes from low-virulence (lentogenic) versus virulent (velogenic) viruses was carried out to identify viral signature motifs that potentially either mimic or complement host miRNA seed sequences. Data show that OAVJ genomes harbor viral seed mimics (vSMs) or viral seed sponges (vSSs) and can mimic host miRNAs or inhibit their regulation of host genes, disrupting cellular pathways. Our analyses showed that velogens encode a statistically significant higher number of vSMs and a lower number of vSSs relative to lentogens. The number of vSMs or vSSs did not correlate with gene length. The analysis of the secondary structures flanking these vSMs and vSSs showed structural features common to miRNA precursors. The inhibition or upregulation of vSS-miR-27b-5p altered P gene expression in a sequence-dependent manner. These data demonstrate that viral transcripts can interact with host miRNAs to alter the outcomes of infection.

Insights and caveats from mining local and global temporal motifs in cryptocurrency transaction networks

Distributed ledger technologies have opened up a wealth of fine-grained transaction data from cryptocurrencies like Bitcoin and Ethereum. This allows research into problems like anomaly detection, anti-money laundering, pattern mining and activity clustering (where data from traditional currencies is rarely available). The formalism of temporal networks offers a natural way of representing this data and offers access to a wealth of metrics and models. However, the large scale of the data presents a challenge using standard graph analysis techniques. We use temporal motifs to analyse two Bitcoin datasets and one NFT dataset, using sequences of three transactions and up to three users. We show that the commonly used technique of simply counting temporal motifs over all users and all time can give misleading conclusions. Here we also study the motifs contributed by each user and discover that the motif distribution is heavy-tailed and that the key players have diverse motif signatures. We study the motifs that occur in different time periods and find events and anomalous activity that cannot be seen just by a count on the whole dataset. Studying motif completion time reveals dynamics driven by human behaviour as well as algorithmic behaviour.

Biosynthesis of Nonribosomal Peptides Chitinimides Reveal a Special Type of Thioesterase Domains.

Non-ribosomal peptide synthetases (NRPSs) and their tailored enzymes have diverse biological functions. In this study, we investigated the biosynthesis and function of chitinimides, which belong to the non-ribosomal peptide (NRP) subfamily featuring a pyrrolidine-containing part (X part) connected to the polypeptide chain via an ester bond. A conserved gene cassette, chmHIJK, is responsible for oxyacylation of the pyrrolidine moiety in the X part. The thioesterase (TE) domain of ChmC (ChmC-TE) catalyzes transesterification reactions with a free X part or methanol as a nucleophilic reagent to form different chitinimides. The crucial amino acid residues in the ChmC-TE domains responsible for the specific recognition of the X part were identified, and they were conserved in all the biosynthetic pathways of this NRP subfamily to form a signature motif, YNHNR, suggesting a special type of TE domain in NRPSs. Chitinimides demonstrate the biological function of promoting the swarming ability of the native producer. This study provides deep insights into the biosynthesis of this special NRP subfamily, and shows that the special TE domain could be used to generate diverse NRPs by combinatorial biosynthesis.

Proteome-scale structural prediction of the giant Marseillevirus reveals conserved folds and putative homologs of the hypothetical proteins.

A significant proportion of the highly divergent and novel proteins of giant viruses are termed "hypothetical" due to the absence of detectable homologous sequences in the existing databases. The quality of genome and proteome annotations often relies on the identification of signature sequences and motifs in order to assign putative functions to the gene products. These annotations serve as the first set of information for researchers to develop workable hypotheses for further experimental research. The structure-function relationship of proteins suggests that proteins with similar functions may also exhibit similar folding patterns. Here, we report the first proteome-wide structure prediction of the giant Marseillevirus. We use AlphaFold-predicted structures and their comparative analysis with the experimental structures in the PDB database to preliminarily annotate the viral proteins. Our work highlights the conservation of structural folds in proteins with highly divergent sequences and reveals potentially paralogous relationships among them. We also provide evidence for gene duplication and fusion as contributing factors to giant viral genome expansion and evolution. With the easily accessible AlphaFold and other advanced bioinformatics tools for high-confidence de novo structure prediction, we propose a combined sequence and predicted-structure-based proteome annotation approach for the initial characterization of novel and complex organisms or viruses.

Developmental and housekeeping transcriptional programs display distinct modes of enhancer-enhancer cooperativity in Drosophila

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and about a million enhancer pairs toward housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are super-additive and combine multiplicatively. Super-additivity between developmental enhancers is promiscuous and neither depends on the enhancers’ endogenous genomic contexts nor on specific transcription factor motif signatures. However, it can be further boosted by Twist and Trl motifs and saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene regulation in complex multi-enhancer developmental loci and genomically clustered housekeeping genes, providing a rationale to interpret the transcriptional impact of non-coding mutations at different loci.

Insights into the functional properties of thioredoxin domain-containing protein 12 (TXNDC12): Antioxidant activity, immunological expression, and wound-healing effect in yellowtail clownfish (Amphiprion clarkii)

Thioredoxin domain-containing protein 12 (TXNDC12) is a member of the thioredoxin-like superfamily that contributes to various thiol-dependent metabolic activities in all living organisms. In this research, the TXNDC12 gene from yellowtail clownfish (Amphiprion clarkii) was structurally characterized using in silico tools, assessed for immunological expression, and evaluated for biological activity using recombinant protein and cellular overexpression. The deduced coding sequence of AcTXNDC12 comprised a 522-bp nucleotide, encoding 173 amino acids with a predicted molecular mass of 19.198 kDa. The AcTXNDC12 protein consists of a66WCGAC70 active motif and a170GDEL173 signature. The highest tissue-specific expression of AcTXNDC12 was observed in the brain tissue, with significant modulation observed in the blood and gill tissues following stimulation of polyinosinic: polycytidylic acid, lipopolysaccharides (LPS), and Vibrio harveyi. In functional assays, recombinant AcTXNDC12 protein (rAcTXNDC12) showed insulin disulfide reduction activity, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) decolorization antioxidant capacity, and ferric (Fe3+) reducing antioxidant potential. Additionally, a significant reduction in nitric oxide production was observed in AcTXNDC12-overexpressed RAW 264.7 cells upon LPS stimulation. Furthermore, genes associated with the regulation of oxidative stress, including nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (Cat), peroxiredoxin 1 (Prx1), and ribonucleotide reductase catalytic subunit M1 (Rrm1) were significantly upregulated in fathead minnow cells overexpressing AcTXNDC12 in response to H2O2 treatment. The scratch wound healing assay demonstrated tissue regeneration and cell proliferation ability upon AcTXNDC12 overexpression. Altogether, the current study elucidated the antioxidant activity, immunological importance, and wound-healing effect of the AcTXNDC12 gene in yellowtail clownfish, providing valuable insights for advancing the aquaculture of A. clarkii fish.

Tetraspanin 5 orchestrates resilience to salt stress through the regulation of ion and reactive oxygen species homeostasis in rice.

Tetraspanins (TETs) are integral membrane proteins, characterized by four transmembrane domains and a unique signature motif in their large extracellular loop. They form dynamic supramolecular complexes called tetraspanin-enriched microdomains (TEMs), through interactions with partner proteins. In plants, TETs are involved in development, reproduction and immune responses, but their role in defining abiotic stress responses is largely underexplored. We focused on OsTET5, which is differentially expressed under various abiotic stresses and localizes to both plasma membrane and endoplasmic reticulum. Using overexpression and underexpression transgenic lines we demonstrate that OsTET5 contributes to salinity and drought stress tolerance in rice. OsTET5 can interact with itself in yeast, suggesting homomer formation. Immunoblotting of native PAGE of microsomal fraction enriched from OsTET5-Myc transgenic rice lines revealed multimeric complexes containing OsTET5, suggesting the potential formation of TEM complexes. Transcriptome analysis, coupled with quantitative PCR-based validation, of OsTET5-altered transgenic lines unveiled the differential expression patterns of several stress-responsive genes, as well as those coding for transporters under salt stress. Notably, OsTET5 plays a crucial role in maintaining the ionic equilibrium during salinity stress, particularly by preserving an elevated potassium-to-sodium (K+/Na+) ratio. OsTET5 also regulates reactive oxygen species homeostasis, primarily by modulating the gene expression and activities of antioxidant pathway enzymes and proline accumulation. Our comprehensive investigation underscores the multifaceted role of OsTET5 in rice, accentuating its significance in developmental processes and abiotic stress tolerance. These findings open new avenues for potential strategies aimed at enhancing stress resilience and making valuable contributions to global food security.

Functional validation of mungbean LEA protein coding gene in bacterial expression system confers salt stress tolerance

Mungbean (Vigna radiata R. Wilczek) is a major tropical food grain legume that is widely cultivated in tropical part of the world. Mungbean like other plants, tolerate and survive limited water situation owing to expression of stress associated proteins that offers membrane stability and cell protection. Late Embryogenesis Abundant (LEA) proteins are among the group of low molecular weight proteins, that play diverse roles in stress protection in several species of plants and animals. A LEA protein coding gene VrLEA2 was isolated from mungbean and its role in stress tolerance has been demonstrated using a bacterial expression system. VrLEA2 gene isolated was of size 893 bp and characterized as a group 1 LEA protein based on the sequence signature motif with presence of hydrophilic domain and a characteristic 20-mer conserved amino acids motif. VrLEA2 gene was cloned into a bacterial expression vector, pET 28a (+), transformed into the E.coli BL21 (DE3) cells for recombinant protein expression and subsequently subjected to antibiotic selection with kanamycin. Functional validation of the VrLEA2 for salt stress tolerance with varied concentration of NaCl (0 mM to 600 mM) showed alteration in colony morphology and reduction in the number of colonies in control compared to the transformed cells demonstrating the improved survival rate of cells expressing VrLEA2 protein. These findings indicate the best use of bacterial expression system for functional validation of plant proteins under stressed environments.

Genome-wide identification of phytosulfokine (PSK) peptide family reveals TaPSK1 gene involved in grain development of wheat (Triticum aestivum L.)

BackgroundPhytosulfokine (PSK) functions as a plant peptide growth factor that plays important and diverse roles in plant development and stress responses. Nevertheless, PSKs have not been systematically analyzed in wheat.ResultsA genome-wide comparative analysis of PSK genes in wheat was conducted and 15 TaPSKs were identified and divided into four subgroups in the wheat genome based on sequence similarity. The examination of motif compositions of TaPSK genes revealed the presence of the YIYTQ signature motif in the C-terminus of all TaPSK polypeptide precursors, with a highly conserved feature across different species. Exogenous application of TaPSK peptide promoted root growth in wheat. Quantitative RT-PCR analysis revealed that the TaPSKs exhibited preferential or tissue-specific expression patterns in wheat. In particular, three homologs of the TaPSK1 genes were specifically expressed in grains, with the strongest expression observed in the developing grains at 15 days after anthesis. Compared with wild type, transgenic rice lines overexpressing TaPSK1-A exhibited larger grain size and higher thousand-grain weight. The promoter region and genomic sequence of the wheat TaPSK1-A gene were cloned. Sequence polymorphism showed that five single-nucleotide polymorphisms (SNPs) were identified in the promoter region of TaPSK1-A. A Kompetitive Allele-Specific PCR (KASP) marker was developed for TaPSK1-A based on –806 bp SNP (C/T transition), and two haplotypes, TaPSK1-A-HapI and TaPSK1-A-HapII were detected in 260 wheat accessions collected from different regions. The expression of TaPSK1-A, promoter activity, and thousand-grain weight (TGW) in the TaPSK1-A-HapII haplotype were higher than those in the TaPSK1-A-HapI haplotype. Furthermore, yeast one-hybrid assays revealed the binding of TaNF-YB1 and TaERF39 to the promoter regions of the TaPSK1-A gene, and TaMADS29 could bind to the promoters of TaPSK1-B and TaPSK1-D genes.ConclusionsComparative genome-wide analysis of TaPSK peptide family revealed that the TaPSK1 gene is involved in wheat grain development, and the developed TaPSK1-A-KASP marker could be utilized for marker-assisted selection breeding of wheat.Graphical

Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a “negative cooperativity” mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.

A novel ABCC9 variant in a Greek family with Cantu syndrome affecting multiple generations highlights the functional role of the SUR2B NBD1.

Cantu syndrome (CS) (OMIM #239850) is an autosomal dominant multiorgan system condition, associated with a characteristic facial phenotype, hypertrichosis, and multiple cardiovascular complications. CS is caused by gain-of-function (GOF) variants in KCNJ8 or ABCC9 that encode pore-forming Kir6.1 and regulatory SUR2 subunits of ATP-sensitive potassium (KATP) channels. A novel heterozygous ABCC9 variant, c.2440G>T; p.Gly814Trp, was identified in three individuals from a four generation Greek family. The membrane potential in cells stably expressing hKir6.1 and hSUR2B with p.Gly814Trp was hyperpolarized compared to cells expressing WT channels, and inside-out patch-clamp assays of KATP channels formed with hSUR2B p.Gly814Trp demonstrated a decreased sensitivity to ATP inhibition, confirming a relatively mild GOF effect of this variant. The specific location of the variant reveals an unrecognized functional role of the first glycine in the signature motif of the nucleotide binding domains in ATP-binding cassette (ABC) protein ion channels.

Cumulative phylogenetic, sequence and structural analysis of Insulin superfamily proteins provide unique structure-function insights.

The insulin superfamily proteins (ISPs), in particular, insulin, IGFs and relaxin proteins are key modulators of animal physiology. They are known to have evolved from the same ancestral gene and have diverged into proteins with varied sequences and distinct functions, but maintain a similar structural architecture stabilized by highly conserved disulphide bridges. The recent surge of sequence data and the structures of these proteins prompted a need for a comprehensive analysis, which connects the evolution of these sequences (427 sequences) in the light of available functional and structural information including representative complex structures of ISPs with their cognate receptors. This study reveals (a) unusually high sequence conservation of IGFs (>90 % conservation in 184 sequences) and provides a possible structure-based rationale for such high sequence conservation; (b) provides an updated definition of the receptor-binding signature motif of the functionally diverse relaxin family members (c) provides a probable non-canonical C-peptide cleavage site in a few insulin sequences. The high conservation of IGFs appears to represent a classic case of resistance to sequence diversity exerted by physiologically important interactions with multiple partners. We also propose a probable mechanism for C-peptide cleavage in a few distinct insulin sequences and redefine the receptor-binding signature motif of the relaxin family. Lastly, we provide a basis for minimally modified insulin mutants with potential therapeutic application, inspired by concomitant changes observed in other insulin superfamily protein members supported by molecular dynamics simulation.

Exploring mechanisms of mupirocin resistance and hyper-resistance.

Mupirocin is a broad-spectrum antibiotic that acts predominantly against Gram-positive bacteria. It is produced by Pseudomonas fluorescens NCIMB 10586 and has been clinically used to treat primary and secondary skin infections and to eradicate nasal colonisation of methicillin-resistant Staphylococcus aureus strains. Mupirocin inhibits protein synthesis by blocking the active site of isoleucyl-tRNA synthetase (IleRS), which prevents the enzyme from binding isoleucine and ATP for Ile-tRNAIle synthesis. Two types of IleRS are found in bacteria - while IleRS1 is susceptible to mupirocin inhibition, IleRS2 provides resistance to cells. These two types belong to distinct evolutionary clades which likely emerged from an early gene duplication in bacteria. Resistance in IleRS2 is based on the loss of interactions that govern mupirocin binding to IleRS1, such as hydrogen bonding to the carboxylate moiety of mupirocin. IleRS2 enzymes with Ki in the millimolar range have recently been discovered. These hyper-resistant IleRS2 variants surprisingly have a non-canonical version of the catalytic motif, which serves as a signature motif of class I aminoacyl-tRNA synthetases to which IleRS belongs. The non-canonical motif, in which the 1st and 3rd positions are swapped, is key for hyper-resistance and can be accommodated without abolishing enzyme activity in IleRS2 but not in IleRS1. Clinical use of mupirocin led to the emergence of resistance in S. aureus. Low-level resistance arises by mutations of the housekeeping IleRS1, while high-level resistance develops by the acquisition of the resistant IleRS2 on a plasmid. There is no evidence that hyper-resistant variants have been found in clinical isolates.

Glycan Profile and Sequence Variants of Certified Ricin Reference Material and Other Ricin Samples Yield Unique Molecular Signature Features.

A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the N-glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin's origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants.

Physico-chemical features and functional relevance of tomato rhomboid proteases

In plants, regulated intramembrane proteolysis (RIP) is crucial for proper growth, development, and stress management. Rhomboid proteases (RPs) residing in the membrane play a vital role in orchestrating RIP. Although RPs can be found in most sequenced genomes, tomato rhomboids (SlRPs) have not yet been studied. Using alternative and comprehensive strategies, we found ten SlRPs encoded in the tomato genome. These SlRPs possess signature motifs and transmembrane domains, showing structural similarity to other members of the RP family. Also, SlRPs are genetically related to other known RPs of the Solanaceae family. Seven of the SlRPs retain serine-histidine catalytic dyads, making them proteolytically active, while three iRhoms lack the dyad and other structural motifs. Although SlRPs could have functional redundancy, their distribution and expression pattern indicate tissue specificity and responsiveness to specific external stimuli. The presence of development and stress-response-related cis-elements in the promoters of SlRPs supports this view. Furthermore, our strategically designed substrate-reporter assay shows that SlRPs have proteolytic activity similar to that of known RPs. This study provides a detailed understanding of all SlRPs and their physico-chemical features, shedding light on their involvement in physiological processes.

The N-terminal signature motif on the transporter MCT1 is critical for CD147-mediated trafficking

The human Solute Carrier (SLC) family member, monocarboxylate transporter 1 (MCT1), transports lactic and pyruvic acid across biological membranes to regulate cellular pH and metabolism. Proper trafficking of MCT1 from the endoplasmic reticulum to the plasma membrane hinges on its interactions with the membrane-bound chaperone protein, CD147. Here, using AlphaFold2 modeling and copurification, we show how a conserved signature motif located in the flexible N-terminus of MCT1 is a crucial region of interaction between MCT1 and the C-terminus of CD147. Mutations to this motif-namely, the thymic cancer linked G19C and the highly conserved W20A-destabilize the MCT1-CD147 complex and lead to a loss of proper membrane localization and cellular substrate flux. Notably, the monomeric stability of MCT1 remains unaffected in mutants, thus supporting the role of CD147 in mediating the trafficking of the heterocomplex. Using the auxiliary chaperone, GP70, we demonstrated that W20A-MCT1 can be trafficked to the plasma membrane, while G19C-MCT1 remains internalized. Overall, our findings underscore the critical role of the MCT1 transmembrane one signature motif for engaging CD147 and identify altered chaperone binding mechanisms between the CD147 and GP70 glycoprotein chaperones.

Characterization of extrachromosomal circular DNAs in plasma of patients with clear cell renal cell carcinoma.

Extrachromosomal circular DNAs (eccDNAs) have been recognized for their significant involvement in numerous biological processes. Nonetheless, the existence and molecular characteristics of eccDNA in the peripheral blood of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have not yet been reported. Our aim was to identify potentially marked plasma eccDNAs in ccRCC patients. The detection of plasma eccDNA in ccRCC patients and healthy controls was performed using the Tn5-tagmentation and next-generation sequencing (NGS) method. Comparisons were made between ccRCC patients and healthy controls regarding the distribution of length, gene annotation, pattern of junctional nucleotide motif, and expression pattern of plasma eccDNA. We found 8,568 and 8,150 plasma eccDNAs in ccRCC patients and healthy controls, respectively. There were no statistical differences in the length distribution, gene annotation, and motif signature of plasma eccDNAs between the two groups. A total of 701 differentially expressed plasma eccDNAs were identified, and 25 plasma eccDNAs with potential diagnostic value for ccRCC have been successfully screened. These up-regulated plasma eccDNAs also be indicated to originate from the genomic region of the tumor-associated genes. This work demonstrates the characterization of plasma eccDNAs in ccRCC and suggests that the up-regulated plasma eccDNAs could be considered as a promising non-invasive biomarker in ccRCC.

Cytochrome P450 enzymes in the black-spotted frog (Pelophylax nigromaculatus): molecular characterization and upregulation of expression by sulfamethoxazole.

Cytochrome P450 (CYP) enzymes are crucial for the detoxification of xenobiotics, cellular metabolism, and homeostasis. This study investigated the molecular characterization of CYP enzymes in the black-spotted frog, Pelophylax nigromaculatus, and examined the regulation of CYP expression in response to chronic exposure to the antibiotic sulfamethoxazole (SMX) at various environmental concentrations (0, 1, 10, and 100μg/L). The full-length cDNA of Pn-CYP26B1 was identified. The sequence included open reading frames of 1,536 bp, encoding proteins comprising 511 amino acids. The signature motif, FxxGxxxCxG, was highly conserved when compared with a number of selected animal species. SMX significantly upregulated the expression of the protein CYP26B1 in frog livers at concentrations of 1 and 10μg/L. SMX showed an affinity for CYP26B1 of -7.6kcal/mol, indicating a potential mechanism for SMX detoxification or adaptation of the frog. These findings contributed to our understanding of the environmental impact of antibiotics on amphibian species and underscored the importance of CYP enzymes in maintaining biochemical homeostasis under exposure to xenobiotic stress.

Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.