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Nucleic Acids Research Sep 2023An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying...
An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying mechanism remains unclear. Although wild-type SOX17 was incapable of inducing pluripotency, SOX17FNV outperformed SOX2 in mouse and human pluripotency reprogramming. In embryonic stem cells, SOX17FNV could replace SOX2 to maintain pluripotency despite considerable sequence differences and upregulated genes expressed in cleavage-stage embryos. Mechanistically, SOX17FNV co-bound OCT4 more cooperatively than SOX2 in the context of the canonical SoxOct DNA element. SOX2, SOX17, and SOX17FNV were all able to bind nucleosome core particles in vitro, which is a prerequisite for pioneer transcription factors. Experiments using purified proteins and in cellular contexts showed that SOX17 variants phase-separated more efficiently than SOX2, suggesting an enhanced ability to self-organise. Systematic deletion analyses showed that the N-terminus of SOX17FNV was dispensable for its reprogramming activity. However, the C-terminus encodes essential domains indicating multivalent interactions that drive transactivation and reprogramming. We defined a minimal SOX17FNV (miniSOX) that can support reprogramming with high activity, reducing the payload of reprogramming cassettes. This study uncovers the mechanisms behind SOX17FNV-induced pluripotency and establishes engineered SOX factors as powerful cell engineering tools.
Topics: Humans; Mice; Animals; Cellular Reprogramming; Transcription Factors; Embryonic Stem Cells; DNA; Point Mutation; Octamer Transcription Factor-3; SOXB1 Transcription Factors; Cell Differentiation; Induced Pluripotent Stem Cells; SOXF Transcription Factors
PubMed: 37607832
DOI: 10.1093/nar/gkad597 -
Research Square Dec 2023Some protein binding pairs exhibit extreme specificities that functionally insulate them from homologs. Such pairs evolve mostly by accumulating single-point mutations,...
Some protein binding pairs exhibit extreme specificities that functionally insulate them from homologs. Such pairs evolve mostly by accumulating single-point mutations, and mutants are selected if their affinity exceeds the threshold required for function. Thus, homologous and high-specificity binding pairs bring to light an evolutionary conundrum: how does a new specificity evolve while maintaining the required affinity in each intermediate? Until now, a fully functional single-mutation path that connects two orthogonal pairs has only been described where the pairs were mutationally close thus enabling experimental enumeration of all intermediates. We present an atomistic and graph-theoretical framework for discovering low molecular strain single-mutation paths that connect two extant pairs, enabling enumeration beyond experimental capability. We apply it to two orthogonal bacterial colicin endonuclease-immunity pairs separated by 17 interface mutations. We were not able to find a strain-free and functional path in the sequence space defined by the two extant pairs. But including mutations that bridge amino acids that cannot be exchanged through single-nucleotide mutations led us to a strain-free 19-mutation trajectory that is completely viable . Our experiments show that the specificity switch is remarkably abrupt, resulting from only one radical mutation on each partner. Furthermore, each of the critical specificity-switch mutations increases fitness, demonstrating that functional divergence could be driven by positive Darwinian selection. These results reveal how even radical functional changes in an epistatic fitness landscape may evolve.
PubMed: 37131620
DOI: 10.21203/rs.3.rs-2836905/v1 -
Function (Oxford, England) 2023Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive expression enhances survival and proliferation of cancer cells, and adversely impacts...
Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34 cells. These results highlight a role of TRPM7 kinase in AKT-driven expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.
Topics: Humans; Proto-Oncogene Proteins c-akt; Cyclooxygenase 2; TRPM Cation Channels; Leukocytes, Mononuclear; Leukemia, Myeloid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Inflammation; Protein Serine-Threonine Kinases
PubMed: 37786778
DOI: 10.1093/function/zqad053 -
Cureus Nov 2023Thyroid cancer, being the prevailing form of endocrine malignancy, exhibits a notable surge in its incidence rates. Follicular thyroid carcinoma (FTC) and papillary... (Review)
Review
Thyroid cancer, being the prevailing form of endocrine malignancy, exhibits a notable surge in its incidence rates. Follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) represent the predominant well-differentiated subtypes and are recognized as the most prevalent forms of thyroid carcinomas. Over the course of several years, numerous molecular, genetic, and epigenetic modifications have been discerned in diverse forms of thyroid neoplasms. Common occurrences comprise point mutations in the and genes, along with chromosomal rearrangements involving the and genes. Thyroid carcinoma, encompassing both FTC and PTC, has been documented in individuals diagnosed with Carney complex (CNC), a hereditary syndrome passed down in an autosomal dominant manner causing increased susceptibility to diverse neoplasms. CNC manifests as a result of inactivating mutations occurring within the tumor-suppressor gene known as , which is responsible for encoding the regulatory subunit of protein kinase A (PKA) type 1α. Studies have shown that this mutation leads to activation of PKA, which, in turn, can induce FTC. In this comprehensive review, we aim to elucidate the intricate molecular mechanisms underlying thyroid tumorigenesis, specifically focusing on the deleterious consequences resulting from the deactivation of the gene.
PubMed: 38074042
DOI: 10.7759/cureus.48503 -
Pathology Oncology Research : POR 2023BCR-ABL1 kinase domain (KD) mutations can lead to resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Here,...
BCR-ABL1 kinase domain (KD) mutations can lead to resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Here, we present the first report of the spectrum of mutations in the BCR-ABL1 KD of CML patients from Azerbaijan. Samples for mutation screening were obtained from patients experiencing resistance to first line TKIs or from patients in acceleration phase (AP) or blast crisis (BC) at the time of diagnosis. The cDNA region corresponding to BCR-ABL1 KD was sequenced by pyrosequencing method. The χ test was used to assess the association of categorical variables between mutation-positive and -negative groups. In addition, the Kaplan-Meier method was applied to generate survival curves. Eight different point mutations were identified in 22 (13.4%) out of 163 CML patients experiencing resistance to TKIs. The types of mutations detected were as follows: Contact binding site mutations 50% (11), SH2 domain mutations 27.4% (six), P-loop mutations 18.1% (four), and SH3 domain mutations accounting for 4.5% (one). The most common mutation was T315I, accounting for 5% ( = 8) of all patients. Significant association was identified between BCR-ABL1 mutations and additional chromosomal aberrations as well as between the mutations and disease phases ( < 0.05). Twelve out of 22 patients with BCR-ABL1 mutations and seven out of eight with T315I were in BC. Overall survival (OS) of the patients with BCR-ABL1 mutations was significantly lower comparing to the patients with no mutation ( < 0.05) and 8 patients with T315I mutation presented OS of 0%. T315I was the most commonly identified BCR-ABL1 mutation in TKI-resistant CML patients of Azerbaijani origin, being associated with disease progression and poor OS.
Topics: Humans; Blast Crisis; Chromosome Aberrations; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation
PubMed: 38188610
DOI: 10.3389/pore.2023.1611518 -
Journal of Virology Jan 2024The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory...
The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin (mACE2) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2 mice, we generated and cloned a more virulent virus. A single isolate (SARS2) was prepared for detailed studies. In 7-11-month-old mACE2 mice, a 10 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.
Topics: Animals; Humans; Mice; 5' Untranslated Regions; Angiotensin-Converting Enzyme 2; COVID-19; Disease Models, Animal; Mice, Inbred C57BL; Nucleotides; Peptidyl-Dipeptidase A; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 38168680
DOI: 10.1128/jvi.01510-23 -
Clinical and Translational... Aug 2023Patients with serrated polyposis syndrome (SPS) have an increased risk to develop colorectal cancer (CRC). Due to an abundance of serrated polyps, these CRCs are assumed...
INTRODUCTION
Patients with serrated polyposis syndrome (SPS) have an increased risk to develop colorectal cancer (CRC). Due to an abundance of serrated polyps, these CRCs are assumed to arise mainly through the serrated neoplasia pathway rather than through the classical adenoma-carcinoma pathway. We aimed to evaluate the pathogenetic routes of CRCs in patients with SPS.
METHODS
We collected endoscopy and pathology data on CRCs and polyps of patients with SPS under treatment in our center. Our primary end point was the proportion of BRAFV600E mutated CRCs, indicating serrated pathway CRCs (sCRCs). CRCs lacking BRAFV600E most likely inferred a classical adenoma-carcinoma origin (aCRCs). We assessed patient, polyp, and CRC characteristics and stratified for BRAFV600E mutation status.
RESULTS
Thirty-five patients with SPS harbored a total of 43 CRCs. Twenty-one CRCs (48.8%) carried a BRAFV600E mutation, 10 of which lacked MLH1 staining and 17 (81%) were located in the proximal colon. Twenty-two CRCs (51.1%) did not carry a BRAFV600E mutation and were MLH1 proficient. Of these 22 putatively aCRCs, 17 (77.3%) were located distally and one-third (36.4%) harbored a pathogenic KRAS or NRAS mutation. In patients with BRAFwt -CRCs, a higher ratio of the median number of conventional adenomas versus serrated polyps was found (4 vs 13) than patients with BRAFV600E -CRCs (1 vs 14).
DISCUSSION
Our study indicates that in patients with SPS, the ratio of sCRCs:aCRCs on average is 50:50. This elevated sCRC:aCRC ratio in patients with SPS, when compared with non-SPS patients, correlates well with the differences in the ratios of the numbers of sessile serrated lesions and conventional adenomas in patients with SPS and non-SPS patients, respectively.
Topics: Humans; Colonic Polyps; Colorectal Neoplasms; Adenomatous Polyposis Coli; Adenoma; Carcinoma
PubMed: 37352472
DOI: 10.14309/ctg.0000000000000611 -
PLoS Genetics Nov 2023PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and...
PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.
Topics: Male; Mice; Animals; Piwi-Interacting RNA; Testis; RNA, Small Interfering; Spermatogenesis; Proteins; Mice, Knockout; Arginine; Argonaute Proteins; Mammals
PubMed: 37956204
DOI: 10.1371/journal.pgen.1011031 -
Frontiers in Immunology 2023The evolution of novel SARS-CoV-2 variants significantly affects vaccine effectiveness. While these effects can only be studied retrospectively, neutralizing antibody...
INTRODUCTION
The evolution of novel SARS-CoV-2 variants significantly affects vaccine effectiveness. While these effects can only be studied retrospectively, neutralizing antibody titers are most used as correlates of protection. However, studies assessing neutralizing antibody titers often show heterogeneous data.
METHODS
To address this, we investigated assay variance and identified virus infection time and dose as factors affecting assay robustness. We next measured neutralization against Omicron sub-variants in cohorts with hybrid or vaccine induced immunity, identifying a gradient of immune escape potential. To evaluate the effect of individual mutations on this immune escape potential of Omicron variants, we systematically assessed the effect of each individual mutation specific to Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5.
RESULTS
We cloned a library of pseudo-viruses expressing spikes with single point mutations, and subjected it to pooled sera from vaccinated hosts, thereby identifying multiple mutations that independently affect neutralization potency.
DISCUSSION
These data might help to predict antigenic features of novel viral variants carrying these mutations and support the development of broad monoclonal antibodies.
Topics: Humans; Retrospective Studies; SARS-CoV-2; COVID-19; Mutation; Vaccination; Antibodies, Neutralizing
PubMed: 38022629
DOI: 10.3389/fimmu.2023.1288794 -
Molecular Metabolism Oct 2023Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic...
OBJECTIVE
Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed.
METHODS
We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R→A).
RESULTS
We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.
CONCLUSIONS
The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.
Topics: Animals; Mice; Diet; Homeostasis; Neuropeptides; Peptide Fragments; Peptide Hormones; Energy Metabolism
PubMed: 37482186
DOI: 10.1016/j.molmet.2023.101781