Mutant Variants Research Articles

Human recombinant tyrosinase destabilization caused by the double mutation R217Q/R402Q.

Oculocutaneous albinism is an autosomal recessive inherited disorder associated with mutations in the TYR gene. A single missense change in the tyrosinase (Tyr) could result in partial or complete loss of catalytic activity. The effect of two genetic mutations in the same Tyr as the molecule is less studied. Here, we study single mutation variants, R217Q, R402Q, and a double mutant variant, R217Q/R402Q, to establish a link between alterations at the level of the atomic model of the protein and the disease phenotype. Human recombinant intra-melanosomal Tyr domains of Tyr and three mutant variants were expressed in T. ni. Larvae were purified using the combination of IMAC and SEC, and diphenolase activities were measured. The Tyr homology model was equilibrated using 100 ns molecular dynamics and analyzed using computational methods. The purified R217Q and R217Q/R402Q variants show decreased catalytic activities compared to those of the Tyr and R402Q variants. The R217Q/R402Q variant has the lowest protein activity and is significantly reduced.

Concordance between human epidermal growth factor receptor 2 (HER2) status by immunohistochemistry (IHC) and next-generation sequencing (NGS) in tissue and blood samples from gastric and gastroesophageal junction cancers (GC/GEJC).

478 Background: An estimated 18% of patients (pts) with GC/GEJC have HER2-positive (HER2+; IHC 3+ or 2+/in situ hybridization–positive [ISH+]) tumors. Although IHC/ISH are the standard testing methods for determining HER2 positivity, NGS can identify HER2 ( ERBB2 ) amplification alongside other biomarkers. This study assessed the concordance of detecting HER2 amplification by tissue/plasma NGS with IHC/ISH HER2 status in pts with GC/GEJC. Methods: Pts were retrospectively identified in the Tempus database. The primary analysis assessed tissue and plasma NGS HER2 copy number (CN) concordance with a HER2+ (IHC 3+ or 2+/ISH+) or HER2− (IHC 0, 1+, or 2+/ISH−) status; NGS samples were collected in ≤30 days of the IHC sample collection date. HER2 amplification was defined as tissue CN ≥8 or plasma log2(CN) ≥0.5; additional amplifications were captured by manual inspection. A secondary analysis assessed the correlation between paired tissue and plasma HER2 log2(CN) in pts with plasma and tissue NGS samples collected ≤30 days apart. The secondary analysis included pts irrespective of IHC sample collection date. Results: Of 1578 pts with GC/GEJC and a curated IHC score evaluated in the primary analysis, 1507 and 305 pts underwent tissue and plasma NGS testing, respectively. Among pts with tissue and plasma NGS results, 902 (59%) and 201 (66%) had primary tumors of the stomach, respectively; all remaining pts had primary tumors of the cardia.According to IHC/ISH, 229 (15%) and 40 (13%) pts were HER2+ among those with tissue and plasma NGS testing, respectively. Of the pts identified as HER2 amplified by tissue and plasma NGS,95% and 92% were HER2+ by IHC/ISH, respectively. Positive percent agreement (PPA) of HER2 amplification by tissue and plasma NGS with IHC/ISH HER2+ status was 70% and 58%, respectively. Concordance rates by PPA, negative PA (NPA), and overall PA (OPA) are summarized in Table. In the secondary analysis, a positive correlation in NGS HER2 log2(CN) was observed in pts with paired tissue and plasma NGS (mutant variant allele frequency >5%; R=0.85; P<2.2e−16; n=307). Conclusions: Based on PPA, we observed moderate concordance between NGS HER2 amplification detection (plasma or tissue) and standard HER2 IHC assessment. NGS is not sufficient to detect all pts with HER2+ GC/GEJC. Further testing is warranted to determine if NGS testing could complement IHC/ISH in identifying pts who may benefit from HER2-directed therapies. IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH− (HER2−), n Total Total 229 1120 Tissue HER2 amplified, n 160 8 168 Tissue HER2 not amplified, n 69 1112 1181 PPA: 69.9% NPA: 99.3% OPA: 94.3% IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH − (HER2 − ), n Total Total 40 233 Plasma HER2 amplified, n 23 2 25 Plasma HER2 not amplified, n 17 231 248 PPA: 57.5% NPA: 99.1% OPA: 93.0%

Safety, efficacy, and on-treatment circulating tumor DNA (ctDNA) changes from a phase 1 study of RMC-6236, a RAS(ON) multi-selective, tri-complex inhibitor, in patients with RAS mutant pancreatic ductal adenocarcinoma (PDAC).

722 Background: PDAC is the third leading cause of cancer mortality, with limited treatment options. Even with multi-agent chemotherapy, patients with second-line (2L) PDAC have a median progression-free survival (PFS) of ≈2–3.5 months and median overall survival (OS) of ≈6–7 months. RAS mutations occur in >90% of patients with PDAC, mainly KRAS G12X (G12X = non-synonymous mutations in KRAS codon 12 [G12]). RMC-6236 is an oral, RAS(ON), multi-selective, tri-complex inhibitor of the active GTP-bound state of both mutant and wild-type RAS. In a Phase 1 study, RMC-6236 demonstrated efficacy and manageable safety in patients with PDAC harboring KRAS G12X or other RAS mutations (NCT05379985). Data on ctDNA reduction with targeted therapy in PDAC are sparse. We report safety, updated efficacy, and exploratory analyses of early ctDNA reduction with clinically active doses of RMC-6236 in patients with RAS mutant PDAC. Methods: Escalating doses of RMC-6236 were administered orally to patients with previously treated RAS mutant PDAC. Additional patients were enrolled for dose optimization and expansion. Patients receiving clinically active doses (160–300 mg QD) of RMC-6236 ≥14 weeks before the July 23, 2024 data cutoff were included. Plasma samples were collected for ctDNA analysis of change in RAS mutant variant allele fraction at baseline (BL; cycle 1, day 1 [C1D1]), and on treatment (C2D1 or C3D1). Results: As of July 23, 2024, 127 patients with RAS mutant PDAC had received RMC-6236 160–300 mg QD. The most common (≥10% of patients) any-grade treatment-related adverse events were rash (91%), diarrhea (48%), nausea (43%), vomiting (31%), stomatitis (31%), fatigue (20%), paronychia (13%), mucosal inflammation (13%), decreased appetite (11%), and peripheral edema (10%). The Table shows objective response rate (ORR), PFS, and OS with 2L RMC-6236. Paired plasma samples were tested in 106/127 patients. Of these, 68 patients with RAS mutant allele ctDNA at BL were evaluable for ctDNA response (Table). Conclusions: RMC-6236 showed a manageable safety profile and encouraging efficacy in patients with previously treated RAS mutant PDAC, and early and deep reductions in RAS mutant ctDNA. RASolute 302, a global, randomized, Phase 3 trial evaluating RMC-6236 as 2L treatment vs chemotherapy in patients with metastatic PDAC, is ongoing. Clinical trial information: NCT05379985 . KRAS G12X RAS mutant Efficacy with 2L RMC-6236 (n=42) (n=57) ORR, % (95% CI)[confirmed + pending confirmation] 29 (16–45) 25 (14–38) Median PFS, months (95% CI) 8.5 (5.3–11.7) 7.6 (5.9–11.1) Median OS, months (95% CI) 14.5 (8.8–not evaluable) 14.5 (8.8–not evaluable) ctDNA Response with 2L+ RMC-6236 (n=56) (n=68) >50% decrease from BL, n (%) 53 (95) 63 (93) 100% decrease from BL, n (%) 28 (50) 32 (47)

Prognostic value of circulating tumor DNA for progression-free survival in patients with locally advanced dMMR/MSI-H colorectal cancer managed without surgery.

256 Background: Immunotherapy has shown the ability to induce a high rate of pathologic and clinical complete response in patients with deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer (CRC). The role of circulating tumor DNA (ctDNA) as a prognostic biomarker for survival in patients who receive non-operative management (NOM) is unknown. Among patients enrolled in a phase II trial (NCT04082572) that evaluated the efficacy of pembrolizumab in patients with locally advanced dMMR/MSI-H solid tumors, we evaluated the impact of ctDNA on progression-free survival (PFS) among 12 patients with locally advanced dMMR/MSI-H CRC who received NOM. Methods: ctDNA testing was performed in the CLIA-certified laboratory at MD Anderson using a 70-gene liquid biopsy panel (LBP-70), which used digital sequencing of cell-free DNA. Formalin-fixed paraffin-embedded tumor samples and germline peripheral blood mononuclear cells were profiled using next-generation sequencing. Mutations in ctDNA were considered tumor-specific if they were confirmed with matched tumor tissue profiling. The lower limit of detection of the LBP-70 assay was a mutational variant allele frequency of <0.3%. "ctDNA clearance" and “ctDNA(-)” status were defined as the absence of any detectable tumor-specific mutations in ctDNA. Two-year progression-free survival (PFS) was estimated using Kaplan-Meier techniques and compared with log-rank tests. Results: The median follow-up between the first cycle of pembrolizumab and last radiographic assessment was approximately three years (36.7 mo; range (range 1.2 – 51.0 mo). Forty-nine LBP-70 assays were performed among the 12 patients over the course of treatment with pembrolizumab. The median number of cycles was 16 (range 1 – 16 cycles). Six patients were ctDNA(-) prior to pembrolizumab, all of whom remained ctDNA(-) after. None of these six patients experienced disease progression (PD) at their last follow-up. The other six patients were ctDNA(+) prior to pembrolizumab (median baseline VAF 0.4%, range 0.3%–4.0%). Of these six patients, three experienced ctDNA clearance while receiving pembrolizumab. None of the three patients who cleared ctDNA experienced PD as of their last follow-up. Of the three patients who did not clear ctDNA, two experienced PD in their primary tumor (at two and six months after their first cycle, respectively), while the third patient remains without PD at their last follow-up. The two-year PFS rate was 100% among the nine patients who were ctDNA(-) after pembrolizumab compared to 33% among the three patients who were ctDNA(+) after pembrolizumab (p = 0.03). Conclusions: ctDNA may be a valuable tool for assessing treatment response and improving the ability to tailor organ-sparing, curative strategies to patients with locally advanced dMMR/MSI-H CRC.

Thermal properties and non-bonded interactions in human PMP2 variants: A molecular dynamics study

Charcot-Marie-Tooth disease has been known for a long time, affecting population worldwide. Many studies on this disease have been conducted, including clinical as well as computational, but no effective medical cure has yet been found. This disease is caused due to mutation in the human peripheral myelin protein 2 (PMP2) responsible for myelin formation and maintenance. In this research work, we have done a comparative molecular dynamics study on the PMP2 protein and its M114T mutant variant, focusing on non-bonded interactions and thermal properties in a biologically relevant environment. The primary objective was to explore the structural differences between the two proteins at temperatures of 305 K, 310 K, and 315 K. Using the NAMD software for molecular dynamics simulations, various analyses were performed, including RMSD estimation, counting number of hydrogen bond formation, salt bridge occupancy assessment, estimation of vdW and electrostatic interaction energies in the system, and estimation of thermal diffusivity of the proteins as well as specific heat capacity (Cv) of the system. The results suggested slight difference between the structure of the two proteins. In the mutant, a slight increase in RMSD values was observed, suggesting a minor reduction in structural stability compared to the wild-type protein. The number of hydrogen bonds formed was generally higher in the wild-type protein, with slightly more differences observed at 305 K and 315 K than at 310 K. Analysis of salt bridge occupancy revealed notable differences in the formation patterns between the two proteins. The mutation was found to have a greater impact on salt bridge formation. In addition to this, the vdW and electrostatic interaction energies were found to be lower in the system with mutant variant, with both energies decreasing gradually as temperature increased, indicating a more robust interaction profile in the system with wild-type protein. Also, the estimated value of thermal diffusivity indicated that the mutant was slightly more efficient at conducting heat than the wild-type protein; the thermal diffusivity of both proteins was much lower than that of water. Finally, a non-linear (concave nature) change in Cv with temperature was observed in both protein systems. The work was further extended to verify the Maxwell-Boltzmann distribution law which confirmed the proper distribution of kinetic energy among particles, and the temperature fluctuation was found to follow a Gaussian distribution during the NVE simulation, which was as expected.

Targeting CRM1 for Progeria Syndrome Therapy.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease caused by progerin, a mutant variant of lamin A. Progerin anchors aberrantly to the nuclear envelope disrupting a plethora of cellular processes, which in turn elicits senescence. We previously showed that the chromosomal region maintenance 1 (CRM1)-driven nuclear export pathway is abnormally enhanced in patient-derived fibroblasts, due to overexpression of CRM1. Interestingly, pharmacological inhibition of CRM1 using leptomycin B rescues the senescent phenotype of HGPS fibroblasts, delineating CRM1 as a potential therapeutic target against HGPS. As a proof of concept, we analyzed the beneficial effects of pharmacologically modulating CRM1 in dermal fibroblasts from HGPS patients and the LMNAG609G/G609G mouse, using the first-in-class selective inhibitor of CRM1 termed selinexor. Remarkably, treatment of HGPS fibroblasts with selinexor mitigated senescence and promoted progerin clearance via autophagy, while at the transcriptional level restored the expression of numerous differentially-expressed genes and rescued cellular processes linked to aging. Invivo, oral administration of selinexor to the progeric mouse resulted in decreased progerin immunostaining in the liver and aorta, decreased progerin levels in most liver, lung and kidney samples analyzed by immunoblotting, and improved aortic histopathology. Collectively our data indicate that selinexor exerts its geroprotective action by at least two mechanisms: normalizing the nucleocytoplasmic partition of proteins with a downstream effect on the aging-associated transcriptome and decreasing progerin levels. Further investigation of the overall effect of selinexor on LmnaG609G/G609G mouse physiology, with emphasis in cardiovascular function is warranted, to determine its therapeutic utility for HGPS and aging-associated disorders characterized by CRM1 overactivity.

Unravelling ATTR Cardiac Amyloidosis in Iceland: A Nationwide Epidemiological Study

Introduction: Cardiac amyloidosis (CA) arises from the deposition of misfolded amyloid proteins in the heart's extracellular matrix, leading to significant cardiac disease. Recent data suggest that CA is under recognised and advances in treatment have increased focus on this condition with the intention of implementing treatment earlier to improve prognosis. There is limited knowledge regarding the prevalence of Transthyretin-CA (ATTR-CA) on a national level, particularly in Iceland. Therefore, this study represents the first nationwide effort to evaluate the baseline characteristics, diagnostics, and treatment of ATTR-CA in Iceland. Methods: A retrospective study of all patients diagnosed with ATTR-CA in Iceland from 6 May 2013 to 11 March 2024 were identified in the Icelandic Cardiac Amyloid Registry (ICE-CAR) created in 2023 by heart failure (HF) specialists at Landspitali University Hospital in Reykjavik, Iceland. Diagnosis was based on different combinations of transthoracic echocardiography (TTE), an elevated Perugini score on bone scintigraphy and heart biopsies, as well measurements of free light chains, M-component measurements in blood and urine. Patients were grouped according to the National Amyloidosis Centre (NAC) prognostic staging system for ATTR-CA. Results: In total, 65 patients with ATTR-CA were identified (males n=60, females n=5, median age 81.4 years [IQR 75.5-85.5 years]), all wild-type and no mutant variant. Diagnosis was made with myocardial biopsy in 7 cases. Upon diagnosis, 83% of the patients had an interventricular septum thickness of ≥15mm and 92% showed a posterior wall thickness of ≥12mm. Approximately 57% of patients belonged to New York Heart Association (NYHA) functional class I-II. The HF phenotypes according to left ventricular ejection fraction (LVEF) were distributed as follows: reduced (HFrEF) n=24 (37%), mildly reduced (HFmrEF) n=9 (14%), preserved (HFpEF) n=29 (44.5%), and unknown LVEF n=3 (4.5%). A total of 54 patients had reported NAC stage: Stage I 23 (35.4%), Stage II 20 (30.8%), Stage III 11 (16.9%). Around 32% received disease modifying treatment. Conclusion Despite the low NYHA class observed in the study population, our findings in Iceland's nationwide assessment of ATTR-CA indicate more advanced age and lower LVEF at diagnosis compared to other studies. This highlights the critical need for early detection and appropriate therapeutic interventions in managing ATTR-CA.

TP53 Mutant Variant Allele Frequency and Cytogenetics Determine Prognostic Groups in MDS/AML for Transplantation.

Results following hematopoietic stem cell transplantation (HSCT) for TP53-mutated myeloid malignancies are disappointing. Several HSCT centers decline to perform HSCT for patients with TP53 mutation because of poor outcomes. In this study, we analyzed 240 patients with TP53-mutated myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) that underwent HSCT. We aimed to identify the patients that benefit most from HSCT. The primary outcome was progression-free survival (PFS). Fifty-two percent of the cohort had AML and the median age of the cohort was 62. AML and MDS outcomes were similar. We identified several favorable prognostic factors for PFS including the absence of complex cytogenetics/5q deletion/7q deletion, a lower variant allele frequency (VAF), a mono-hit status and the use of a matched-related donor. Using classification and regression tree analysis, we identified VAF and cytogenetics as the two most important prognostic factors. Patients with TP53mut VAF ≥ 50% had a 2-year PFS of 3%, patients with TP53mut VAF < 50% and complex/5q/7q cytogenetics abnormalities had 2-year PFS of 22%. Patients with TP53mut VAF < 50% and without complex/5q/7q cytogenetics had 2-year PFS of 60%. This data informs clinical practice and helps patients decide whether to pursue HSCT.

Huntingtin plays an essential role in the adult hippocampus.

The consequences of non-pathogenic huntingtin (HTT) reduction in the mature brain are of substantial importance as clinical trials for numerous HTT-lowering therapies are underway; many of which are non-selective in that they reduce both mutant and wild type protein variants. In this study, we injected CaMKII-promoted AAV-Cre directly into the hippocampus of adult HTT floxed mice to explore the role of wild-type huntingtin (wtHTT) in adult hippocampal pyramidal neurons and the broader implications of its loss. Our findings reveal that wtHTT depletion results in profound macroscopic morphological abnormalities in hippocampal structure, accompanied by significant reactive gliosis. At the synaptic level, we identified a marked reduction in presynaptic terminals 1-2 months following wtHTT loss; this was contrasted by an increased density of postsynaptic mushroom spines and larger amplitudes of spontaneous excitatory postsynaptic currents, indicative of disrupted synaptic homeostasis. Furthermore, intrinsic neuronal excitability was significantly diminished in CA1 pyramidal neurons lacking wtHTT, and we observed a complete loss of NMDA receptor-dependent long-term potentiation. Unexpectedly, synapse density returned to control levels 6-8 months following wtHTT loss, despite the ongoing presence of macroscopic morphological abnormalities, altered anxiety-related behaviors and clear impairments in spatial learning and memory. Overall, these findings uncover a previously unrecognized role of wtHTT as a critical regulator of hippocampal function in the mature brain, and highlight the hippocampus as a potentially vulnerable region to the adverse effects of non-selective HTT reduction.

Conformational Dynamics of Mitochondrial Inorganic Pyrophosphatase hPPA2 and Its Changes Caused by Pathogenic Mutations.

Inorganic pyrophosphatases, or PPases, are ubiquitous enzymes whose activity is necessary for a large number of biosynthetic reactions. The catalytic function of PPases is dependent on certain conformational changes that have been previously characterized based on the comparison of the crystal structures of various complexes. The current work describes the conformational dynamics of a structural model of human mitochondrial pyrophosphatase hPPA2 using molecular dynamics simulation, all-atom principal component analysis, and coarse-grained normal mode analysis. In addition to the wild-type enzyme, four mutant variants of hPPA2 were characterized that correspond to the natural pathogenic variants causing severe mitochondrial dysfunction and cardio pathologies. As a result, we identified the global type of flexible motion that seems to be shared by other dimeric PPases. This motion is discussed in terms of the allosteric behavior of the protein. Analysis of the observed conformational dynamics revealed the formation of a binding site for anionic ligands in the active site that could be relevant to enzyme catalysis. Based on the comparison of the wild-type and mutant PPases dynamics, we suggest the possible molecular mechanisms of the functional incompetence of hPPA2 caused by mutations. The results of this work allow for deeper insight into the structural basis of PPase function and the possible effects of pathogenic mutations on the protein structure and function.

Neutralising and Non-neutralising Antibodies to SARS-CoV-2: Role during Infection and in the Evolution of Antigenic structure

Relevance. COVID-19, caused by the SARS-CoV-2 virus, remains a global public health threat despite the end of the pandemic. In the four years since the onset of the pandemic, the SARS-CoV-2 genome has undergone significant changes, particularly in the gene encoding the spike (S) protein. These changes resulted from the accumulation of immune responses in the human population, allowing the virus to evade the immune response. A significant proportion of the population was infected early in the pandemic or vaccinated with vaccines based on earlier variants of the virus. The emergence of new mutant variants raises concerns about the potential for severe COVID-19 in previously infected or vaccinated individuals.Аim. To examine the specifics of antibody formation, as well as the spectrum and functional activity of these antibodies in patients with COVID-19.Conclusions. Antibodies produced in response to infection or vaccination show diversity in spectrum and functional activity. Changes in the viral genome may reduce antibody effectiveness, highlighting the importance of monitoring new SARS-CoV-2 variants and developing adapted vaccines. These data will be key in shaping COVID-19 vaccination and treatment strategies in a changing epidemiological situation.

Exome sequencing reveals a rare damaging variant in GRIN2C in familial late-onset Alzheimer's disease

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disorder with both genetic and environmental factors contributing to its pathogenesis. While early-onset AD has well-established genetic determinants, the genetic basis for late-onset AD remains less clear. This study investigates a large Italian family with late-onset autosomal dominant AD, identifying a novel rare missense variant in GRIN2C gene associated with the disease, and evaluates the functional impact of this variant.MethodsAffected and unaffected members from a Northern Italian family were included. Genomic DNA from family members was extracted and initially screened for pathogenic mutations in APP, PSEN1, and PSEN2, and screened for 77 genes associated with neurodegenerative conditions using NeuroX array assay. Exome sequencing was performed on three affected individuals and two healthy relatives. Bioinformatics analyses were conducted. Functional analysis was performed using primary neuronal cultures, and the impact of the variant was assessed through immunocytochemistry and electrophysiology.ResultsPathogenic variants were not identified in APP, PSEN1, or PSEN2, nor in the 77 genes in NeuroX array assay. Exome Sequencing revealed the c.3215C > T p.(A1072V) variant in GRIN2C gene (NM 000835.6), encoding for the glutamate ionotropic receptor N-methyl-D-aspartate receptor (NMDA) type subunit 2C (GluN2C). This variant segregated in 6 available AD patients in the family and was absent in 9 healthy relatives. Primary rat hippocampal neurons overexpressing GluN2CA1072V showed an increase in NMDAR-induced currents, suggesting altered glutamatergic transmission. Surface expression assays demonstrated an elevated surface/total ratio of the mutant GluN2C, correlating with the increased NMDAR current. Additionally, immunocytochemistry revealed in neurons expressing the mutant variant a reduced colocalization between the GluN2C subunit and 14-3-3 proteins, which are known to facilitate membrane trafficking of NMDARs.DiscussionWe identified a rare missense variant in GRIN2C associated with late-onset autosomal dominant Alzheimer's disease. These findings highlight the role of GluN2C-containing NMDARs in glutamatergic signaling and their potential contribution to AD pathogenesis.

Differential Inhibition by Cenobamate of Canonical Human Nav1.5 Ion Channels and Several Point Mutants.

Cenobamate is a new and highly effective antiseizure compound used for the treatment of adults with focal onset seizures and particularly for epilepsy resistant to other antiepileptic drugs. It acts on multiple targets, as it is a positive allosteric activator of γ-aminobutyric acid type A (GABAA) receptors and an inhibitor of neuronal sodium channels, particularly of the late or persistent Na+ current. We recently evidenced the inhibitory effects of cenobamate on the peak and late current component of the human cardiac isoform hNav1.5. The determined apparent IC50 values of 87.6 µM (peak) and 46.5 µM (late current) are within a clinically relevant range of concentrations (the maximal plasma therapeutic effective concentration for a daily dose of 400 mg in humans is 170 µM). In this study, we built a 3D model of the canonical hNav1.5 channel (UniProt Q14524-1) in open conformation using AlphaFold2, embedded it in a DPPC lipid bilayer, corrected the residue protonation state (pH 7.2) with H++, and added 2 Na+ ions in the selectivity filter. By molecular docking, we found the cenobamate binding site in the central cavity. We identified 10-point mutant variants in the binding site region and explored them via docking and MD. Mutants N1462K/Y (rs1064795922, rs199473614) and M1765R (rs752476527) (by docking) and N932S (rs2061582195) (by MD) featured higher predicted affinity than wild-type.

Thermodynamic binding properties of a novel umami octapeptide K1ADEDSLA8 and its mutational variants p.A2G, p.D5E, and p.A2G + p.D5E (BMP) in complex with the umami receptor hT1R1/hT1R3

Thermodynamic binding properties of a novel umami octapeptide K1ADEDSLA8 and its mutational variants p.A2G, p.D5E, and p.A2G + p.D5E (BMP) in complex with the umami receptor hT1R1/hT1R3

Yeast Dnm1G178R causes altered organelle dynamics and sheds light on the human DRP1G149R disease mechanism.

Mitochondrial morphology is a result of regulated opposite events called fission and fusion and requires the GTPase, dynamin-related protein 1 (DRP1/Dnm1), or its homologs. A recent clinical report identified a heterozygous missense mutation in the human DRP1 that replaces Glycine (G) 149 with Arginine (R) and results in debilitating conditions in the patient. In this study, we mimicked this mutation in yeast Dnm1 (G178R) and investigated the impact of the pathogenic mutation on the protein's function. We provide evidence that the substitution of G with R in the G3 motif of the GTPase domain, renders the protein non-functional and in a dominant-negative way. The mutation hampers the distribution, localization, and function of the protein. Cells expressing the mutant variant exhibit a block in mitochondrial fission and altered peroxisome morphology and number.

Molecular profiling of BRCA1/BRCA2 gene variants in Pakistani breast cancer patients.

To determine the frequency of genetic variants in breast cancer types 1 and 2 gene in breast cancer Pakistani patients. The case-control study was conducted at the Islamic International Medical College and Pakistan Railway Hospital, Rawalpindi, Pakistan, from October 2022 to August 2023, and comprised females with breast cancer in group A, with an age range of 23-83 years (mean 51.9 ± 10.8 years) and as many healthy, age-matched female controls in group B, with age range of 26-82 years(mean 57.8 ±10.1 years). Peripheral blood samples were taken from all the participants. Allele-specific tetra amplification refractory mutation system-polymerase chain reaction was used to determine the frequency of c.4485G>A, c.4508C>A, c.5278G>C and c.5503C>T genetic variants in breast cancer type 1 gene, and c.92G>A and c.3109C>T genetic variants in breast cancer type 2 gene. Data was analysed using SPSS 27. Of the 336 female participants 168(50%) were in each of the 2 groups. None of the group B controls had genetic variants in breast cancer type 1 and 2 genes. In group A patients, 4(2.3%) had the variant c.4485G>A in breast cancer type 1 gene, while other breast cancer type 1 variants were negative. Also, 9(5.3%) group A patients had variants c.92G>A and c.3109C>T in breast cancer type 2 gene. Cost-effective, polymerase chain reaction-based genetic testing could effectively identify mutant variants of breast cancer types 1 and 2 genes in the Pakistani population.

Predictive Modeling of Gene Expression and Localization of DNA Binding Site Using Deep Convolutional Neural Networks.

Despite the sequencing revolution, large swaths of the genomes sequenced to date lack any information about the arrangement of transcription factor binding sites on regulatory DNA. Massively Parallel Reporter Assays (MPRAs) have the potential to dramatically accelerate our genomic annotations by making it possible to measure the gene expression levels driven by thousands of mutational variants of a regulatory region. However, the interpretation of such data often assumes that each base pair in a regulatory sequence contributes independently to gene expression. To enable the analysis of this data in a manner that accounts for possible correlations between distant bases along a regulatory sequence, we developed the Deep learning Adaptable Regulatory Sequence Identifier (DARSI). This convolutional neural network leverages MPRA data to predict gene expression levels directly from raw regulatory DNA sequences. By harnessing this predictive capacity, DARSI systematically identifies transcription factor binding sites within regulatory regions at single-base pair resolution. To validate its predictions, we benchmarked DARSI against curated databases, confirming its accuracy in predicting transcription factor binding sites. Additionally, DARSI predicted novel unmapped binding sites, paving the way for future experimental efforts to confirm the existence of these binding sites and to identify the transcription factors that target those sites. Thus, by automating and improving the annotation of regulatory regions, DARSI generates experimentally actionable predictions that can feed iterations of the theory-experiment cycle aimed at reaching a predictive understanding of transcriptional control.

Dissecting the Kaiso binding profile in clear renal cancer cells

BackgroundThere has been a notable increase in interest in the transcriptional regulator Kaiso, which has been linked to the regulation of clonal hematopoiesis, myelodysplastic syndrome, and tumorigenesis. Nevertheless, there are no consistent data on the binding sites of Kaiso in vivo in the genome. Previous ChIP-seq analyses for Kaiso contradicted the accumulated data of Kaiso binding sites obtained in vitro. Here, we studied this discrepancy by characterizing the distribution profile of Kaiso binding sites in Caki-1 cells using Kaiso-deficient cells as a negative control, and compared its pattern on chromatin with that in lymphoblastoid cell lines.ResultsWe employed Caki-1 kidney carcinoma cells and their derivative, which lacks the Kaiso gene, as a model system to identify the genomic targets of Kaiso. The principal binding motifs for Kaiso are CGCG and CTGCNAT, with 60% of all binding sites containing both sequences. The significance of methyl-DNA binding activity was confirmed through examination of the genomic distribution of the E535A mutant variant of Kaiso, which cannot bind methylated DNA in vitro but is able to interact with CTGCNA sequences. Our findings indicate that Kaiso is present at CpG islands with a preference for methylated ones. We identified Kaiso target genes whose methylation and transcription are dependent on its expression. Furthermore, Kaiso binding sites are enriched at CpG islands, with partial methylation at the 5' and/or 3' boundaries. We discovered CpG islands exhibiting wave-like methylation patterns, with Kaiso detected in the majority of these areas. Similar data were obtained in other cell lines.ConclusionThe present study delineates the genomic distribution of Kaiso in cancer cells, confirming its role as a factor with a complex mode of DNA binding and a strong association with CpG islands, particularly with methylated and eroded CpG islands, revealing a new potential Kaiso target gene—SQSTM1, involved in differentiation of acute myeloid leukemia cells. Furthermore, we discovered the existence of a new class of CpG islands characterized by wave-like DNA methylation.

PD-1 interactome in osteosarcoma: identification of a novel PD-1/AXL interaction conserved between humans and dogs

The PD-1/PDL-1 immune checkpoint inhibitors revolutionized cancer treatment, yet osteosarcoma remains a therapeutic challenge. In some types of cancer, PD-1 receptor is not solely expressed by immune cells but also by cancer cells, acting either as a tumor suppressor or promoter. While well-characterized in immune cells, little is known about the role and interactome of the PD-1 pathway in cancer. We investigated PD-1 expression in human osteosarcoma cells and studied PD-1 protein–protein interactions in cancer. Using U2OS cells as a model, we confirmed PD-1 expression by western blotting and characterized its intracellular as well as surface localization through flow cytometry and immunofluorescence. High-throughput analysis of PD-1 interacting proteins was performed using a pull-down assay and quantitative mass spectrometry proteomic analysis. For validation and molecular modeling, we selected tyrosine kinase receptor AXL—a recently reported cancer therapeutic target. We confirmed the PD-1/AXL interaction by immunoblotting and proximity ligation assay (PLA). Molecular dynamics (MD) simulations uncovered binding affinities and domain-specific interactions between extracellular (ECD) and intracellular (ICD) domains of PD-1 and AXL. ECD complexes exhibited strong binding affinity, further increasing for the ICD complexes, emphasizing the role of ICDs in the interaction. PD-1 phosphorylation mutant variants (Y223F and Y248F) did not disrupt the interaction but displayed varying strengths and binding affinities. Using bemcentinib, a selective AXL inhibitor, we observed reduced binding affinity in the PD-1/AXL interaction, although it was not abrogated. To facilitate the future translation of this finding into clinical application, we sought to validate the interaction in canine osteosarcoma. Osteosarcoma spontaneously occurs at significantly higher frequency in dogs and shares close genetic and pathological similarities with humans. We confirmed endogenous expression of PD-1 and AXL in canine osteosarcoma cells, with PD-1/AXL interaction preserved in the dog cells. Also, the interacting residues remain conserved in both species, indicating an important biological function of the interaction. Our study shed light on the molecular basis of the PD-1/AXL interaction with the implication for its conservation across species, providing a foundation for future research aimed at improving immunotherapy strategies and developing novel therapeutic approaches.

Adaptive Laboratory Evolution of Flavin Functionality Identifies Dihydrolipoyl Dehydrogenase as One of the Critical Points for the Activity of 7,8-Didemethyl-Riboflavin as a Surrogate for Riboflavin in Escherichia coli.

Riboflavin analogs lacking one methyl group (7α or 8α) can still serve as a surrogate for riboflavin in riboflavin-deficient microorganisms or animals. The absence of both methyl groups at once completely abolishes this substitution capability. To elucidate the molecular mechanisms behind this phenomenon, we performed an adaptive laboratory evolution experiment (in triplicate) on an E. coli strain auxotrophic for riboflavin. As a result, the riboflavin requirement of the E. coli strain was reduced ~10-fold in the presence of 7,8-didemethyl-riboflavin. The whole genome sequencing of E. coli strains isolated from three experiments revealed two mutation hotspots: lpdA coding for the flavoenzyme dihydrolipoyl dehydrogenase (LpdA), and ompF coding for the major outer membrane protein. In order to investigate the essentiality of flavin's methyl groups to LpdA, the wild type and mutant variants of lpdA were cloned. At least two lpdA mutants increased the fitness of E. coli, and when 7,8-didemethyl-flavin was added to the growth medium, the increase was significant. To the best of our knowledge, an adaptive laboratory evolution experiment running in triplicate as a tool for the identification of mutation hotspots in the genome of microorganisms exposed to metabolic stress challenges is described here for the first time.