Sequence Motifs Research Articles

Molecular cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos.

Embryonic genome activation (EGA) is a critical step in early embryonic development, as it marks the transition from relying on maternal factors to the initiation of transcription from embryo's own genome. The factors associated with EGA are not well understood and need further investigation. PRD-like (PRDL) homeodomain transcription factors (TFs) are considered to play crucial roles in this early event during development but these TFs have evolved differently, even within mammalian lineages. Different numbers of PRDL TFs have been predicted in bovine (Bos taurus); however, their divergent evolution requires species-specific confirmation and functional investigations. In this study, we conducted molecular cloning of mRNAs for the PRDL TFs ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 in bovine oocytes or in vitro fertilized (IVF) preimplantation embryos. Our results confirmed the expression of PRDL TF genes in early bovine development at the cDNA level and uncovered their structures. For each investigated PRDL TF gene, we isolated at least one homeodomain-encoding cDNA fragment, indicative of DNA binding and thus potential role in transcriptional regulation in developing bovine embryos. Additionally, our cDNA cloning approach allowed us to reveal breed-related differences in bovine, as evidenced by the identification of a high number of single nucleotide variants (SNVs) across the PRDL class homeobox genes. Subsequently, we observed the prediction of the 9aa transactivation domain (9aaTAD) motif in the putative protein sequence of TPRX3 leading us to conduct functional analysis of this gene. We demonstrated that the TPRX3 overexpression in bovine fibroblast induces not only protein-coding genes but also short noncoding RNAs involved in splicing and RNA editing. We supported this finding by identifying a shared set of genes between our and published bovine early embryo development datasets. Providing full-length cDNA evidence for previously predicted homeobox genes that belong to PRDL class improves the annotation of the bovine genome. Updating the annotation with seven developmentally-important genes will enhance the accuracy of RNAseq analysis with datasets derived from bovine preimplantation embryos. In addition, the absence of TPRX3 in humans highlights the species-specific and TF-specific regulation of biological processes during early embryo development.

Correlating Mechanical Properties and Sequence Motifs in Artificial Spider Silk by Targeted Motif Substitution.

The major ampullate silk of orb-weaving spiders is renowned for its exceptional mechanical properties, including high tensile strength and extensibility. The development of artificial spider silk presents a promising alternative to traditional fibers with significant environmental impacts. This study aims to elucidate the relationship between sequence motifs of natural spider silk and the mechanical properties of artificial spider silk. Using the Spider Silkome Database, we identified motifs correlated with specific physical properties and substituted them into MaSp2-based mini-spidroin BP1. We then measured the mechanical properties of the resulting recombinant artificial spider silk through tensile tests, observed structural properties via birefringence measurement and wide-angle X-ray scattering, and evaluated the water response through boiled water shrinkage tests. Introducing a positively correlated motif increased the tensile strength by 9.3%, while a negatively correlated motif decreased it by 5.1%, confirming the sequence-property relationship. These findings demonstrate that targeted motif substitution can effectively control the physical properties of artificial spider silk, facilitating the development of sustainable biomaterials with tailored mechanical properties for diverse industrial applications.

Structure-aware annotation of leucine-rich repeat domains.

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains.

DmTGS: Precise Targeted Enrichment Long-Read Sequencing Panel for Tandem Repeat Detection.

Tandem repeats (TRs) are abundant in the human genome and associated with repeat expansion disorders. Our study aimed to develop a tandem repeat panel utilizing targeted long-read sequencing to evaluate known TRs associated with these disorders and assess its clinical utility. We developed a targeted long-read sequencing panel for 70 TR loci, termed dynamic mutation third-generation sequencing (dmTGS), using the PacBio Sequel II platform. We tested 108 samples with suspected repeat expansion disorders and compared the results with conventional molecular methods. For 108 samples, dmTGS achieved an average of 8000 high-fidelity reads per sample, with a mean read length of 4.7 kb and read quality of 99.9%. dmTGS outperformed repeat-primed-PCR and fluorescence amplicon length analysis-PCR in distinguishing expanded from normal alleles and accurately quantifying repeat counts. The method demonstrated high concordance with confirmatory methods (rlinear = 0.991, P < 0.01), and detected mosaicism with sensitivities of 1% for FMR1 CGG premutation and 5% for full mutations. dmTGS successfully identified interruptive motifs in genes that conventional methods had missed. For variable number TRs in the PLIN4 gene, dmTGS identified precise repeat counts and sequence motifs. Screening 57 patients with suspected genetic muscular diseases, dmTGS confirmed repeat expansions in genes such as GIPC1, NOTCH2NLC, NUTM2B-AS1/LOC642361, and DMPK. Additionally, dmTGS detected CCG interruptions in CTG repeats in 8 myotonic dystrophy type 1 patients with detailed characterization. dmTGS accurately detects repeat sizes and interruption motifs associated with repeat expansion disorders and demonstrates superior performance compared to conventional molecular methods.

Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.

Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed bysequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.

Deciphering Mechanisms of Adipocyte Differentiation in Abdominal Fat of Broilers.

The excessive deposition of abdominal fat tissue (AFT) in broilers has emerged as a major concern in the poultry industry. Despite some progress in recent years, the molecular mechanisms underlying AFT development remain ambiguous. The current study combined RNA-seq with transposase-accessible chromatin sequencing (ATAC-seq) to map the dynamic profiling of chromatin accessibility and transcriptional reprogramming in AFT adipocyte differentiation in broilers at day 3 (D3) and D14. Our results found that the levels of CDK1 and PCNA were down-regulated at D14, D28, and D42 compared to D3, while the levels of C/EBPα and FABP4 were up-regulated at D14 and D42 compared to D3. Meanwhile, PPARγ was significantly up-regulated at D28 and D42. RNA-seq of AFT identified 1705 up-regulated and 1112 down-regulated differential expression genes (DEGs) between D3 and D14. Pathways based on up-regulated DEGs mainly enriched some pathways related to adipocyte differentiation, while down-regulated DEGs pointed to DNA replication, cell cycle, and gap junction. Gene set enrichment analysis (GSEA) revealed that DNA replication and the cell cycle were down-regulated at D14, while the insulin signaling pathway was up-regulated. In the OA-induced immortalized chicken preadipocyte (ICP2) model, protein dynamic changes were consistent with AFT from D3 to D14. Same pathways were enriched in ICP2. In addition, based on overlapped DEGs from AFT and ICP2, enriched pathways related to adipocyte differentiation or proliferation mentioned above were all involved. A total of 1600 gain and 16727 loss differential peaks (DPs) were identified in ICP2 by ATAC-seq. Predicted genes from DPs at the promoter regions were enriched in glycerophospholipid metabolism, TGF-β signaling, FoxO signaling, and ubiquitin-mediated proteolysis. DNA motifs predicted 159 transcription factors (TFs) based on gain and loss peaks from the promoter regions, where 1 and 10 TFs were overlapped with up or down TFs from DEGs. Overall, this study presents a framework for the comprehension of the epigenetic regulatory mechanisms of adipocyte differentiation and identifies candidate genes and potential TFs involved in AFT adipocyte differentiation in broilers.

Biogenesis of Cytochromes c and c1 in the Electron Transport Chain of Malaria Parasites.

Plasmodium malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and is a key antimalarial drug target. ETC function requires cytochromes c and c1, which are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate the biogenesis of the mature cytochrome c or c1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, Plasmodium parasites are like yeast and encode two separate HCCS homologues thought to be specific for heme attachment to cyt c (HCCS) or cyt c1 (HCC1S). To test the function and specificity of Plasmodium falciparum HCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c1 biogenesis and caused lethal ETC dysfunction that was not reversed by the overexpression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in Escherichia coli of cyt c hemylation by P. falciparum HCCS and HCC1S strongly suggest that both homologues are essential for mitochondrial ETC function and have distinct specificities for the biogenesis of cyt c and c1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

Genome-wide profiling of soybean WRINKLED1 transcription factor binding sites provides insight into seed storage lipid biosynthesis

Understanding the regulatory mechanisms controlling storage lipid accumulation will inform strategies to enhance seed oil quality and quantity in crop plants. The WRINKLED1 transcription factor (WRI1 TF) is a central regulator of lipid biosynthesis. We characterized the genome-wide binding profile of soybean (Gm)WRI1 and show that the TF directly regulates genes encoding numerous enzymes and proteins in the fatty acid and triacylglycerol biosynthetic pathways. GmWRI1 binds primarily to regions downstream of target gene transcription start sites. We showed that GmWRI1-bound regions are enriched for the canonical WRI1 DNA binding element, the ACTIVATOR of Spomin::LUC1/WRI1 (AW) Box (CNTNGNNNNNNNCG), and another DNA motif, the CNC Box (CNCCNCC). Functional assays showed that both DNA elements mediate transcriptional activation by GmWRI1. We also show that GmWRI1 works in concert with other TFs to establish a regulatory state that promotes fatty acid and triacylglycerol biosynthesis. In particular, comparison of genes targeted directly by GmWRI1 and by GmLEC1, a central regulator of the maturation phase of seed development, reveals that the two TFs act in a positive feedback subcircuit to control fatty acid and triacylglycerol biosynthesis. Together, our results provide unique insights into the genetic circuitry in which GmWRI1 participates to regulate storage lipid accumulation during seed development.

Phosphorylation in the Plasmodium falciparum Proteome: A Meta-Analysis of Publicly Available Data Sets.

Malaria is a deadly disease caused by Apicomplexan parasites of the Plasmodium genus. Several species of the Plasmodium genus are known to be infectious to humans, of which P. falciparum is the most virulent. Post-translational modifications (PTMs) of proteins coordinate cell signaling and hence regulate many biological processes in P. falciparum homeostasis and host infection, of which the most highly studied is phosphorylation. Phosphosites on proteins can be identified by tandem mass spectrometry (MS) performed on enriched samples (phosphoproteomics), followed by downstream computational analyses. We have performed a large-scale meta-analysis of 11 publicly available phosphoproteomics data sets to build a comprehensive atlas of phosphosites in the P. falciparum proteome, using robust pipelines aimed at strict control of false identifications. We identified a total of 26,609 phosphorylated sites on P. falciparum proteins, split across three categories of data reliability (gold/silver/bronze). We identified significant sequence motifs, likely indicative of different groups of kinases responsible for different groups of phosphosites. Conservation analysis identified clusters of phosphoproteins that are highly conserved and others that are evolving faster within the Plasmodium genus, and implicated in different pathways. We were also able to identify over 180,000 phosphosites within Plasmodium species beyond falciparum, based on orthologue mapping. We also explored the structural context of phosphosites, identifying a strong enrichment for phosphosites on fast-evolving (low conservation) intrinsically disordered regions (IDRs) of proteins. In other species, IDRs have been shown to have an important role in modulating protein-protein interactions, particularly in signaling, and thus warranting further study for their roles in host-pathogen interactions. All data have been made available via UniProtKB, PRIDE, and PeptideAtlas, with visualization interfaces for exploring phosphosites in the context of other data on Plasmodium proteins.

Protein language models learn evolutionary statistics of interacting sequence motifs

Protein language models (pLMs) have emerged as potent tools for predicting and designing protein structure and function, and the degree to which these models fundamentally understand the inherent biophysics of protein structure stands as an open question. Motivated by a finding that pLM-based structure predictors erroneously predict nonphysical structures for protein isoforms, we investigated the nature of sequence context needed for contact predictions in the pLM Evolutionary Scale Modeling (ESM-2). We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modeling approaches like Markov Random Fields and Multivariate Gaussian models. We further investigated how ESM-2 "stores" information needed to predict contacts by comparing sequence masking strategies, and found that providing local windows of sequence information allowed ESM-2 to best recover predicted contacts. This suggests that pLMs predict contacts by storing motifs of pairwise contacts. Our investigation highlights the limitations of current pLMs and underscores the importance of understanding the underlying mechanisms of these models.

Deep quantification of substrate turnover defines protease subsite cooperativity.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

Cortex-wide characterization of decision-making neural dynamics during spatial navigation.

Decision-making during freely moving behaviors involves complex interactions among many cortical and subcortical regions. However, the spatiotemporal coordination across regions to generate a decision is less understood. Using a head-mounted widefield microscope, cortex-wide calcium dynamics were recorded in mice expressing GCaMP7f as they navigated an 8-maze using two paradigms. The first was an alternating pattern that required short term memory of the previous trial to make the correct decision and the second after a rule change to a fixed path in which rewards were delivered only on the left side. Identification of cortex-wide activation states revealed differences between the two paradigms. There was a higher probability for a visual/retrosplenial cortical state during the alternating paradigm and higher probability of a secondary motor and posterior parietal state during left-only. Three state sequences (motifs) illustrated both anterior and posterior activity propagations across the cortex. The anterior propagating motifs had the highest probability around the decision and propagating motifs peaked following the decision. The latter, likely reflecting internal feedback to influence future actions, were more common in the left-only paradigm. Therefore, the probabilities and sequences of cortical states differ when working memory is required versus a fixed trajectory reward paradigm.

Genome-wide identification of CAMTA genes and their expression dependence on light and calcium signaling during seedling growth and development in mung bean

BackgroundCalmodulin-binding transcription activator (CAMTA) is comprised of a group of transcription factors and plays an important role in the Ca2+ signaling pathway, mediating various molecular responses via interactions with other transcription factors and binding to the promoter region of specific genes. Mung beans (Vigna radiata) are one of the most commonly consumed commodities in Asia. To date, CAMTA proteins have not been characterized in this important crop plant.ResultsEight paralogous VrCAMTA genes were identified and found to be distributed on five of the 11 chromosomes. The proteins possessed CG-1 DNA-binding domains with bipartite NLS signals, ankyrin domains, CaM-binding IQ motifs, and CaM-binding domain (CaMBD). The 2 kb upstream regions of VrCAMTA genes contained sequence motifs of abscisic acid-responsive elements (ABRE) and ethylene-responsive elements (ERE), and binding sites for transcription factors of the bZIP and bHLH domains. Analysis of RNA-seq data from a public repository revealed ubiquitous expression of the VrCAMTA genes, as VrCAMTA1 was expressed at the highest level in seedling leaves, whereas VrCAMTA8 was expressed at the lowest level, which agreed with the RT-qPCR analysis performed on the first true leaves. On day four after leaf emergence, all VrCAMTA genes were upregulated, with VrCAMTA1 exhibiting the highest degree of upregulation. In darkness on day 4, upregulation was not observed in most VrCAMTA genes, except VrCAMTA7, for which a low degree of upregulation was found, whereas no difference was found in VrCAMTA8 expression between light and dark conditions. Treatment with calcium ionophores enhanced VrCAMTA expression under light and/or dark conditions at different times after leaf emergence, suggesting that calcium signaling is involved in the light-induced upregulation of VrCAMTA gene expression.ConclusionsThe expression dependence of nearly all VrCAMTA genes on light and calcium signaling suggests their possible differential but likely complementary roles during the early stages of mung bean growth and development.

Dissecting the role of transcription factor AP2-M in Babesia asexual replication.

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN_0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases.

Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification.

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.

Binding profiles for 961 Drosophila and C. elegans transcription factors reveal tissue-specific regulatory relationships.

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the efforts of the Model Organism ENCyclopedia Of DNA Elements (modENCODE) and the model organism Encyclopedia of Regulatory Networks (modERN) consortia to systematically assay TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). These datasets comprise 605 TFs identifying 3.6M sites in the fly and 356 TFs identifying 0.9 M sites in the worm and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed "metapeaks", that larger metapeaks have characteristics of high occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single cell RNA-seq data in a machine learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent-daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing GFP-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell type-specific TF-target relationships.

Advancements in long non-coding RNA-based therapies for cancer: targeting, delivery, and clinical implications.

Long non-coding RNAs (lncRNAs) have been in the spotlight for the past two decades due to their extensive role in regulating a wide range of cellular processes. Development, differentiation, regulation, and modulation are some of the vital cellular cascades coordinated by these molecules. Despite their importance, there has been limited literature on their practical implications in cancer prevention. Advancements in lncRNA biology have enabled the characterization of numerous secondary structures and sequence motifs, which could serve as potential targets for cellular therapies. Several studies have highlighted the involvement of lncRNAs in human pathologies, where they can be targeted by small molecules or antisense oligonucleotides to prevent diseases. However, progress has been hindered by the challenge of developing specific delivery vehicles for targeted delivery. Recent improvements in sequence optimization and nucleotide modification have enhanced drug stability and reduced the immunogenicity of lncRNA-based therapies, yet further advances are needed to fully realize their potential in treating complex diseases like cancer. This review aims to explore current lncRNA biology, their mechanisms of action, nanoformulation strategies, and the clinical trials focused on lncRNA delivery systems.

Post-transcriptional methylation of mitochondrial-tRNA differentially contributes to mitochondrial pathology

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N1-methylation of guanosine at position 9 (m1G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m1G9 methylation, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving the structure and activity of the variant. These results have therapeutic implications, suggesting that the N1-methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Abstract A037: Enhanced anti-tumor activity through targeted immunotherapy combined with mutant KRAS inhibition in pancreatic cancer

Abstract Constitutively-active KRASG12D has become targetable via clinical-stage KRASG12D-selective small molecule inhibitors. Prior preclinical research has demonstrated that KRASG12D inhibition makes pancreatic ductal adenocarcinoma (PDAC) more amenable to immunotherapy. However, the impact of such inhibition on antigen presentation by tumor cells remains unclear. To elucidate this phenomenon, we employed a multi-omic profiling workflow to investigate alterations in the proteome and peptide MHC ligandome (immunopeptidome) following treatment of HPAC human cancer cells with a RAS(ON) G12D-selective inhibitor RM-044 (representative of investigational agent RMC-9805) and/or IFNɣ in vitro. Multi-omic profiling revealed significant changes in antigen presentation following KRASG12D inhibition. Pairwise comparisons of differentially expressed proteins in treated vs. untreated conditions showed increased expression of proteins associated with interferon signaling and replication stress. Notably, antigen processing and presentation proteins such as HLA-B and -C alpha chains showed some of the highest expression levels. Immunopeptidomic analysis revealed a 2-fold increase in ligand diversity and abundance in treated vs. untreated conditions. Sequence motif enrichment analysis depicted motifs consistent with HPAC’s HLA-B and -C but not -A alleles, aligning with proteomic findings and highlighting how the integration of these two data layers informs the shaping of the immunopeptidomic landscape. Building on these findings, we hypothesized that KRASG12D signaling inhibition in preclinical models could augment immune responses initiated by immunization targeting tumor antigens in vivo. Our hypothesis was that the release of antigens from KRASG12D-induced cell death, coupled with increased MHC-I expression in remaining tumor cells, would enhance interactions with T cells activated by immunization. To test this, we orthotopically inoculated C57Bl/6 mice with a murine PDAC-KRASG12D cell line engineered to express the model self/tumor-associated antigen gp100. Mice were immunized with either empty ionizable lipid nanoparticles (e-iLNPs) or iLNPs packaged with mRNA encoding full-length gp100 (gp100-iLNP). Following a priming and boost series, mice were treated with either KRASG12D inhibitior MRTX1133 or vehicle for two weeks. Remarkably, mice treated with the gp100 mRNA-iLNP immunization followed by KRASG12D inhibition experienced significant tumor regression compared to mice that received KRASG12D inhibition alone. These preclinical findings underscore the dynamic nature by which PDAC antigen presentation can be modulated by KRAS signaling inhibition and IFNɣ. The combination of allele-specific KRAS inhibition with tumor antigen immunization resulted in significant tumor regression in a stringent in vivo PDAC preclinical model, compared to either treatment modality alone. This rationalizes the combination of both approaches for enhanced anti-tumor activity, offering a strategy that is worth exploring clinically for improving outcomes in pancreatic cancer treatment. Citation Format: Amanda Creech, Hailey Lee, Khalid Rashid, Thuc Le, Alykhan Premji, Tony Luu, Ahmad Kassem, Weihang Zhao, Luyi Li, Nanping Wu, Norbert Pardi, James Wohlschlegel, Timothy Donahue, Caius Radu. Enhanced anti-tumor activity through targeted immunotherapy combined with mutant KRAS inhibition in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A037.