N-terminal Mutant Research Articles

Cebpa is required for haematopoietic stem and progenitor cell generation and maintenance in zebrafish.

The CCAAT enhancer binding protein alpha (CEBPA) is crucial for myeloid differentiation and the balance of haematopoietic stem and progenitor cell (HSPC) quiescence and self-renewal, and its dysfunction can drive leukemogenesis. However, its role in HSPC generation has not been fully elucidated. Here, we utilized various zebrafish cebpa mutants to investigate the function of Cebpa in the HSPC compartment. Co-localization analysis showed that cebpa expression is enriched in nascent HSPCs. Complete loss of Cebpa function resulted in a significant reduction in early HSPC generation and the overall HSPC pool during embryonic haematopoiesis. Interestingly, while myeloid differentiation was impaired in cebpa N-terminal mutants expressing the truncated zP30 protein, the number of HSPCs was not affected, indicating a redundant role of Cebpa P42 and P30 isoforms in HSPC development. Additionally, epistasis experiments confirmed that Cebpa functions downstream of Runx1 to regulate HSPC emergence. Our findings uncover a novel role of Cebpa isoforms in HSPC generation and maintenance, and provide new insights into HSPC development.

Asymmetric Dynamics Drive Catalytic Activation of the Hsp90 Chaperone.

The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended 19F NMR spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to show that within the context of heterodimeric Hsp90, the conformationally dynamic subunit stimulates the ATPase activity of the normal subunit. The contrasting dynamic properties of the subunits within heterodimeric Hsp90 provide a mechanistic framework to understand the molecular basis for asymmetric Hsp90 activation and its importance for the biological function of Hsp90.

Insulin-Degrading Enzyme Efficiently Degrades polyQ Peptides but not Expanded polyQ Huntingtin Fragments.

Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

Improved Thermal Stability of a Novel Acidophilic Phytase.

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.

Understanding a point mutation signature D54K in the caspase activation recruitment domain of NOD1 capitulating concerted immunity via atomistic simulation

Point mutation D54K in the human N-terminal caspase recruitment domain (CARD) of nucleotide-binding oligomerization domain −1 (NOD1) abrogates an imperative downstream interaction with receptor-interacting protein kinase (RIPK2) that entails combating bacterial infections and inflammatory dysfunction. Here, we addressed the molecular details concerning conformational changes and interaction patterns (monomeric-dimeric states) of D54K by signature-based molecular dynamics simulation. Initially, the sequence analysis prioritized D54K as a pathogenic mutation, among other variants, based on a sequence signature. Since the mutation is highly conserved, we derived the distant ortholog to predict the sequence and structural similarity between native and mutant. This analysis showed the utility of 33 communal core residues associated with structural-functional preservation and variations, concurrently served to infer the cryptic hotspots Cys39, Glu53, Asp54, Glu56, Ile57, Leu74, and Lys78 determining the inter helical fold forming homodimers for putative receptor interaction. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural alteration that takes place in the N-terminal mutant CARD where coils changed to helices (45 α3- L4-α4-L6- α683) in contrast to native (45T2-L4-α4-L6-T483). Likewise, the C-terminal helices 93T1-α7105 connected to the loops distorted compared to native 93α6-L7105 may result in conformational misfolding that promotes functional regulation and activation. These structural perturbations of D54K possibly destabilize the flexible adaptation of critical homotypic NOD1CARD-CARDRIPK2 interactions (α4Asp42-Arg488α5 and α6Phe86-Lys471α4) is consistent with earlier experimental reports. Altogether, our findings unveil the conformational plasticity of mutation-dependent immunomodulatory response and may aid in functional validation exploring clinical investigation on CARD-regulated immunotherapies to prevent systemic infection and inflammation. Communicated by Ramaswamy H. Sarma

Two somatic mutations in the androgen receptor N-terminal domain are oncogenic drivers in hepatocellular carcinoma

The androgen receptor (AR) plays an important role in male-dominant hepatocellular carcinoma, and specific acquired somatic mutations of AR have been observed in HCC patients. Our previous research have established the role of AR wild type as one of the key oncogenes in hepatocarcinogenesis. However, the role of hepatic acquired somatic mutations of AR remains unknown. In this study, we identify two crucial acquired somatic mutations, Q62L and E81Q, situated close to the N-terminal activation function domain-1 of AR. These mutations lead to constitutive activation of AR, both independently and synergistically with androgens, making them potent driver oncogene mutations. Mechanistically, these N-terminal AR somatic mutations enhance de novo lipogenesis by activating sterol regulatory element-binding protein-1 and promote glycogen accumulation through glycogen phosphorylase, brain form, thereby disrupting the AMPK pathway and contributing to tumorigenesis. Moreover, the AR mutations show sensitivity to the AMPK activator A769662. Overall, this study establishes the role of these N- terminal hepatic mutations of AR as highly malignant oncogenic drivers in hepatocarcinogenesis and highlights their potential as therapeutic targets for patients harboring these somatic mutations.

Ubiquitin Ligase SCF-FBXO11 Controls a Network of RNA-Binding Proteins and Splicing in MDS

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

Mowat-Wilson syndrome factor ZEB2 controls early formation of human neural crest through BMP signaling modulation

Mowat-Wilson syndrome is caused by mutations in ZEB2, with patients exhibiting characteristics indicative of neural crest (NC) defects. We examined the contribution of ZEB2 to human NC formation using a model based on human embryonic stem cells. We found ZEB2 to be one of the earliest factors expressed in prospective human NC, and knockdown revealed a role for ZEB2 in establishing the NC state while repressing pre-placodal and non-neural ectoderm genes. Examination of ZEB2 N-terminal mutant NC cells demonstrates its requirement for the repression of enhancers in the NC gene network and proper NC cell terminal differentiation into osteoblasts and peripheral neurons and neuroglia. This ZEB2 mutation causes early misexpression of BMP signaling ligands, which can be rescued by the attenuation of BMP. Our findings suggest that ZEB2 regulates early human NC specification by modulating proper BMP signaling and further elaborate the molecular defects underlying Mowat-Wilson syndrome.

TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression

The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPADM AML are GATA2 and TET2, however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPADM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

Investigation of the acetic acid stress response in Saccharomyces cerevisiae with mutated H3 residues.

Enhanced levels of acetic acid reduce the activity of yeast strains employed for industrial fermentation-based applications. Therefore, unraveling the genetic factors underlying the regulation of the tolerance and sensitivity of yeast towards acetic acid is imperative for optimising various industrial processes. In this communication, we have attempted to decipher the acetic acid stress response of the previously reported acetic acid-sensitive histone mutants. Revalidation using spot-test assays and growth curves revealed that five of these mutants, viz., H3K18Q, H3S28A, H3K42Q, H3Q68A, and H3F104A, are most sensitive towards the tested acetic acid concentrations. These mutants demonstrated enhanced acetic acid stress response as evidenced by the increased expression levels of AIF1, reactive oxygen species (ROS) generation, chromatin fragmentation, and aggregated actin cytoskeleton. Additionally, the mutants exhibited active cell wall damage response upon acetic acid treatment, as demonstrated by increased Slt2-phosphorylation and expression of cell wall integrity genes. Interestingly, the mutants demonstrated increased sensitivity to cell wall stress-causing agents. Finally, screening of histone H3 N-terminal tail truncation mutants revealed that the tail truncations exhibit general sensitivity to acetic acid stress. Some of these N-terminal tail truncation mutants viz., H3 [del 1-24], H3 [del 1-28], H3 [del 9-24], and H3 [del 25-36] are also sensitive to cell wall stress agents such as Congo red and caffeine suggesting that their enhanced acetic acid sensitivity may be due to cell wall stress induced by acetic acid.

Targeted activation of human ether-à-go-go-related gene channels rescues electrical instability induced by the R56Q+/- long QT syndrome variant.

Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

Aberrant splicing of mutant huntingtin in Huntington's disease knock-in pigs

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disease caused by a CAG repeat expansion in exon1 of the huntingtin gene (HTT). This expansion leads to the production of N-terminal mutant huntingtin protein (mHtt) that contains an expanded polyglutamine tract, which is toxic to neurons and causes neurodegeneration. While the production of N-terminal mHtt can be mediated by proteolytic cleavage of full-length mHtt, abnormal splicing of exon1-intron1 of mHtt has also been identified in the brains of HD mice and patients. However, the proportion of aberrantly spliced exon1 mHTT in relation to normal mHTT exon remains to be defined. In this study, HTT exon1 production was examined in the HD knock-in (KI) pig model, which more closely recapitulates neuropathology seen in HD patient brains than HD mouse models. The study revealed that aberrant spliced HTT exon1 is also present in the brains of HD pigs, but it is expressed at a much lower level than the normally spliced HTT exon products. These findings suggest that careful consideration is needed when assessing the contribution of aberrantly spliced mHTT exon1 to HD pathogenesis, and further rigorous investigation is required.

Monomerization of TDP-43 is a key determinant for inducing TDP-43 pathology in amyotrophic lateral sclerosis.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization-deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization-deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell-derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Hydrogen bonds connecting the N-terminal region and the DE loop stabilize the monomeric structure of transthyretin.

Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF β-sheets during the unfolding of the TTR monomer by stabilizing the interaction between β-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.

CRISPR-Cas9 correction in the DMD mouse model is accompanied by upregulation of Dp71f protein

Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a deficiency in the dystrophin protein. The most frequent types of disease-causing mutations in the DMD gene are frameshift deletions of one or more exons. Precision genome editing systems such as CRISPR-Cas9 have shown potential to restore open reading frames in numerous animal studies. Here, we applied an AAV-CRISPR double-cut strategy to correct a mutation in the DMD mouse model with exon 8-34 deletion, encompassing the N-terminal actin-binding domain. We report successful excision of the 100-kb genomic sequence, which includes exons 6 and 7, and partial improvement in cardiorespiratory function. While corrected mRNA was abundant in muscle tissues, only a low level of truncated dystrophin was produced, possibly because of protein instability. Furthermore, CRISPR-Cas9-mediated genome editing upregulated the Dp71f dystrophin isoform on the sarcolemma. Given the previously reported Dp71-associated muscle pathology, our results question the applicability of genome editing strategies for some DMD patients with N-terminal mutations. The safety and efficacy of CRISPR-Cas9 constructs require rigorous investigation in patient-specific animal models.

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

Abstract A016: Expanded RAS proteoform landscape in malignant cell lines revealed by top-down mass spectrometry

Abstract The RAS family of GTPases (HRAS, KRAS4A, KRAS4B, NRAS) provide a formidable challenge to standard proteomic workflows. Linking key N-terminal mutations (e.g., G12C, G13D) with C-terminal post-translational modifications (PTMs) on a given RAS isoform by proteolytic digestion and peptide-based analysis is further complicated by their high sequence identity, low endogenous abundance, and labile PTMs. Conversely, top-down mass spectrometry (TDMS), which measures intact and modified protein forms (proteoforms), can precisely identify and confidently localize mutations and PTMs within each RAS isoform sequence. Moreover, by combining immunoprecipitation with TDMS (IP-TDMS), endogenous RAS proteoforms can be isolated to near-homogeneity from malignant cell lines, detected at high signal-to-noise ratios via selected ion monitoring, and targeted for optimized MS2-based characterization to provide molecular detail simply unachievable by other proteomic methods. The NCI RAS Initiative has developed an optimized RAS proteoform assay incorporating IP-TDMS analysis on an Orbitrap Fusion Lumos mass spectrometer. We initially employed recombinant versions of each RAS isoform to determine the optimal targeted MS2 fragmentation (tMS2) parameters for sequence characterization and localization of endogenous PTMs. We next isolated FLAG-tagged KRAS4B -WT, -G12D, and -G12C expression constructs from HeLa cells to refine our liquid chromatography and tMS2 parameters while targeting stoichiometrically abundant KRAS4B proteoforms within each population. We then isolated FLAG-tagged HRAS-, KRAS4A-, KRAS4B-, and NRAS-G12V proteins from HeLa or Panc1 cells for further refinement of isoform-specific tMS2 parameters with emphasis on capturing unusual or labile PTMs (e.g. palmitoylation). This allowed us to visualize and directly compare the most abundant RAS isoform-specific proteoform populations within these cellular contexts. Complementary peptide-based analyses were performed on all samples to validate RAS isoform-specific PTM landscapes and facilitate detection of their respective endogenous versions. Finally, we applied our optimized IP-TDMS workflow to a panel of well-characterized malignant and RAS mutant cell lines with the goal of expanding our knowledge of the endogenous RAS proteoform landscape. In doing so, we identified a wealth of novel HRAS, KRAS4A, KRAS4B, and NRAS proteoforms bearing stoichiometrically abundant PTMs differing markedly from the established literature. We also performed direct comparisons between isoform- and mutation-specific RAS proteoforms, revealing clear indications of context dependence. These initial results further underscore the potential for top-down proteomics to broaden our understanding of RAS-dependent signaling within a range of cancer contexts. They also reveal that extensive investigations remain to be performed to elucidate the roles of RAS PTMs and proteoforms in normal and oncogenic signaling. Citation Format: Caroline J. DeHart, Robert A. D'Ippolito, Kanika Sharma, Nicole Fer, Brian Smith, Mackenzie Meyer, Scott Eury, Abigail Neish, Katie Powell, Vanessa Wall, William Burgan, Dominic Esposito, Anna E. Maciag, Frank McCormick, Dwight V. Nissley. Expanded RAS proteoform landscape in malignant cell lines revealed by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A016.

Specific Mutations in APC, with Prognostic Implications in Metastatic Colorectal Cancer.

Loss-of-function mutations in the adenomatous polyposis coli (APC) gene are common in metastatic colorectal cancer (mCRC). However, the characteristic of APC specific mutations in mCRC is poorly understood. Here, we explored the clinical and molecular characteristics of N-terminal and C-terminal side APC mutations in Chinese patients with mCRC. Hybrid capture-based next-generation sequencing was performed on tumor tissues from 275 mCRC pati-ents to detect mutations in 639 tumor-associated genes. The prognostic value and gene-pathway difference between APC specific mutations in mCRC patients were analyzed. APC mutations were highly clustered, accounting for 73% of all mCRC patients, and most of them were truncating mutations. The tumor mutation burden of the N-terminal side APC mutations group (n=76) was significantly lower than that of the C-terminal side group (n=123) (p < 0.001), further confirmed by the public database. Survival analysis showed that mCRC patients with N-terminus side APC mutations had longer overall survival than C-terminus side. Tumor gene pathway analysis showed that gene mutations in the RTK/RAS, Wnt and transforming growth factor β signaling pathways of the C-terminal group were significantly higher than those of the N-terminal group (p < 0.05). Additionally, KRAS, AMER1, TGFBR2, and ARID1A driver mutations were more common in patients with C-terminal side APC mutations. APC specific mutations have potential function as mCRC prognostic biomarkers. There are obvious differences in the gene mutation patterns between the C-terminus and N-terminus APC mutations group, which may have certain guiding significance for the subsequent precise treatment of mCRC.

N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates

BackgroundArgonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested.ResultsOur in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates.ConclusionsMany other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.