Mutation Of Residues Research Articles

Enhanced reactive oxygen species and metabolism are involved in the reduction of tribenuron-methyl residues in Tartary buckwheat mutants

Tartary buckwheat, known for its rapid growth, short growth cycles, and adaptability, is cultivated worldwide, particularly in East Asia and Eastern Europe. However, weed infestations severely impact yield and quality, and the absence of effective herbicides poses a considerable challenge to Tartary buckwheat production. To address this, we used ethyl methane sulfonate (EMS) mutagenesis to create a mutant population of Tartary buckwheat seeds. We applied a targeted screening process using the herbicide tribenuron-methyl (TM) to select mutants with reduced sensitivity to the herbicide. Integrative analysis of transcriptomic and metabolomic data indicated that TM primarily inhibited photosynthesis and amino acid biosynthesis pathways in sensitive plants, leading to toxicity. Conversely, resistant plants could reduce the toxic effects of TM on Tartary buckwheat by increasing antioxidant enzyme activity and enhancing secondary metabolic pathways such as flavonoid biosynthesis. TM also upregulated the expression of a gene encoding uridine diphosphate glucuronic acid transferase-like (FtUGT79L). Overexpression of FtUGT79L in Arabidopsis substantially increased total flavonoid content and improved the resistance of Arabidopsis to TM at the seedling and adult stages. The study provides insights into innovative approaches for breeding herbicide-resistant Tartary buckwheat germplasm and serves as an important reference for the development of herbicide-resistant varieties of other crops.

Exploration of urease-aided calcium carbonate mineralization by enzyme analyses of Neobacillus mesonae strain NS-6.

Urease containing nickel cofactor is crucial for urea-hydrolytic induced calcium carbonate (CaCO3) precipitation (UICP). However, limited information exists regarding the influence of amino acid residues interacting with nickel ions in its structure on induced CaCO3 mineralization. Herein, RT-qPCR was used to demonstrate that the addition of NiCl2 dramatically upregulated the expression of urease structural gene ureC that was correlated with nickel binding in Neobacillus mesonae strain NS-6. Homology modeling and molecular docking were employed to construct the three-dimensional structure of urease and seek the key residues involved in nickel binding process, and virtual mutation technology was adopted to inform three key residues coordinated with nickel ions and urea, His249, His275, and Asp363. Four metrics, including root mean square deviation values for mutations of those key residues in urease-urea complexes severally and wild-type, were calculated by molecular dynamics simulations when they were mutated into alanine, respectively. Subsequently, the mutations of H249A, H275A, and D363A were characterized using western blotting to reveal a decrease in the relative expression and activity of urease, along with a corresponding reduction in CaCO3 precipitation. Ultimately, the mutations also exhibited that they had lower substrate affinity and catalytic efficiency for urea through enzymatic properties analysis. The findings suggested that those residues played a pivotal role in UICP of strain NS-6, which would expand the theoretical basis for modulating urease activity.IMPORTANCEUrease-producing bacterium is of great importance in diverse application fields, such as environmental remediation, due to its key driving characteristics in catalyzing urea hydrolysis via urea-hydrolytic induced CaCO3 precipitation (UICP). As essential cofactors of urease, nickel ions play a crucial role in regulating urease catalysis and maintaining structural stability. Numerous investigations have emphasized the impact of nickel ions on urease activity in recent years, to our best knowledge, only a few literatures have studied the molecular-level regulation of nickel-ligand residues. This study focused on the highly urease-producing bacterial Neobacillus mesonae NS-6 to explore the effects of specific nickel-ligand residues on the urease-aided CaCO3 mineralization process using molecular simulation predictions and targeted mutation experiments. The aim was to provide a molecular-level understanding of the interactive effects between urea and critical residues associated with the urease active center, as well as propose an effective modification strategy to enhance the application of UICP in future environmental areas.

Highly multiplexed design of an allosteric transcription factor to sense new ligands

Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures – four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone – as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors.

High fitness paths can connect proteins with low sequence overlap.

The structure and function of a protein are determined by its amino acid sequence. While random mutations change a protein's sequence, evolutionary forces shape its structural fold and biological activity. Studies have shown that neutral networks can connect a local region of sequence space by single residue mutations that preserve viability. However, the larger-scale connectedness of protein morphospace remains poorly understood. Recent advances in artificial intelligence have enabled us to computationally predict a protein's structure and quantify its functional plausibility. Here we build on these tools to develop an algorithm that generates viable paths between distantly related extant protein pairs. The intermediate sequences in these paths differ by single residue changes over subsequent steps - substitutions, insertions and deletions are admissible moves. Their fitness is evaluated using the protein language model ESM2, and maintained as high as possible subject to the constraints of the traversal. We document the qualitative variation across paths generated between progressively divergent protein pairs, some of which do not even acquire the same structural fold. The ease of interpolating between two sequences could be used as a proxy for the likelihood of homology between them.

CSIG-31. ANNEXIN A7 ALTERNATIVE SPLICING IN GLIOMA STEM CELLS FACILITATES THE DEGRADATION OF EGFR BY PHOSPHORYLATING TYROSINE RESIDUES EXCLUSIVE TO PROTEIN ISOFORM 1

Abstract The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling and resulting in hyperactive RTK signaling. Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates in PDX glioma stem cells (GSCs). We demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. JX14 GSCs were made to overexpress ANXA7-I1 with lentivirus to conduct immunofluorescence experiments measuring EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway for the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (R2=0.9267, P<0.0001). Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). On the other hand, ANXA7 siRNA KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1 to prefer recycling again while EV cells were unaffected. I1 overexpression significantly reduced EGFR signaling through degradation, which is driven by the phosphorylation of I1-exclusive tyrosine residues in phosphomimetic (p<0.01) and non-phosphorylatable protein mutants. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for GSCs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.

Conformational plasticity links structural instability of NAA10F128I and NAA10F128L mutants to their catalytic deregulation

The acetylation of proteins' N-terminal amino groups by the N-acetyltransferase complexes plays a crucial role in modulating the spatial stability and functional activities of diverse human proteins. Mutations disrupting the stability and function of NAA10 result in X-linked rare genetic disorders. In this study, we conducted a global analysis of the impact of fifteen disease-associated missense mutations in NAA10. The analyses revealed that mutations in specific residues, such as Y43, V107, V111, and F128, predictably disrupted interactions essential for NAA10 stability, while most mutations (except R79C, A111W, Q129P, and N178K) expectedly led to structural destabilization. Mutations in many conserved residues within short linear motifs and post-translational modification sites were predicted to affect NAA10 functionality and regulation. All mutations were classified as pathogenic, with F128I and F128L identified as the most destabilizing mutations. The findings show that the F128L and F128I mutations employ different mechanisms for the loss of catalytic activities of NAA10F128L and NAA10F128I due to their structural instability. These two mutations induce distinct folding energy states that differentially modulate the structures of different regions of NAA10F128L and NAA10F128I. Specifically, the predicted instability caused by the F128I mutation results in decreased flexibility within the substrate-binding region, impairing the substrate peptide binding ability of NAA10F128I. Conversely, F128L is predicted to reduce the flexibility of the region containing the acetyl-CoA binding residues in NAA10F128L. Our study provides insights into the mechanism of catalytic inactivation of mutants of NAA10, particularly elucidating the mechanistic features of the structural and functional pathogenicity of the F128L and F128I mutations.

Functional dynamics of human ubiquitin C-terminal hydrolases

Ubiquitin C-terminal hydrolases (UCHs) are crucial enzymes within the ubiquitin-proteasome system, characterized by a characteristic Gordian knotted topology. Another important structural feature of the UCH family is a hydrophobic β-sheet core containing a conserved catalytic triad of cysteine, histidine, and aspartate wrapped by several α-helices and a crossover loop. The catalytic triad cleaves the (iso) peptide bond at the C-terminus of ubiquitin via a nucleophilic attack. The highly dynamic crossover loop is involved in substrate binding and selectivity. UCHs play vital roles in various cellular processes, such as cell signaling, DNA repair, neuroprotection, and tumor suppression. Point mutations in catalytic and non-catalytic residues of UCHs are linked to various diseases, including cancers and neurodegeneration. Additionally, post-translational modifications (PTMs), such as oxidation, impact the deubiquitinase activity of UCHs and increase aggregation propensity. This review focuses on how disease-associated point mutations, PTMs, and interactions with different binding partners modulate the structural and functional dynamics of UCHs and how perturbations of these functional dynamics are characterized using a battery of biophysical techniques to gain insights into the molecular mechanisms underlying UCH dysfunction and diseases.

Allosteric modulation of the Lon protease via ssDNA binding and local charge changes.

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduce ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements, we find this change in activity correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

Mitochondrial inhibitors reveal roles of specific respiratory chain complexes in CRY-dependent degradation of TIM

Drosophila Cryptochrome (CRY) is an essential photoreceptor that mediates the resetting of the circadian clock by light. in vitro studies demonstrated a critical role of redox cycling of the FAD cofactor for CRY activation by light. However, it is unknown if CRY responds to cellular redox environment to modulate the circadian clock. We report here that the mitochondrial respiratory chain impinges on CRY activity. Inhibition of complex III and V blocks CRY-mediated degradation of TIMELESS (TIM) in response to light, and also blocks light-induced CRY degradation. On the other hand, inhibition of complex I facilitates TIM degradation even in the dark. Mutations of critical residues of the CRY C-terminus promote TIM degradation in the dark, even in the presence of complex III and V inhibitors. We propose that complex III and V activities are important for activation of CRY in response to light. Interestingly, we found that transcriptional repressor functions of Drosophila and mammalian CRY proteins are not affected by mitochondrial inhibitors. Together these data suggest that the two functions of CRY have different sensitivity to disruptions of the mitochondrial respiratory chain: one is sensitive to mitochondrial activities that enable resetting, the other is insensitive so as to sustain the molecular oscillator.

Revealing SARS-CoV-2 Mpro mutation cold and hot spots: Dynamic residue network analysis meets machine learning

Deciphering the effect of evolutionary mutations of viruses and predicting future mutations is crucial for designing long-lasting and effective drugs. While understanding the impact of current mutations on protein drug targets is feasible, predicting future mutations due to natural evolution of viruses and environmental pressures remains challenging. Here, we leveraged existing mutation data during the evolution of the SARS-CoV-2 protein drug target main protease (Mpro) to test the predictive power of dynamic residue network (DRN) analysis in identifying mutation cold and hot spots. We conducted molecular dynamics simulations on the Mpro of SARS-CoV-2 (Wuhan strain) and calculated eight DRN metrics (averaged BC, CC, DC, EC, ECC, KC, L, PR), each of which identifies a unique network feature within the protein. The sets of residues with the highest and lowest values for each metric, comprising potential cold and hot spots, were compared to published biochemical analyses and per residue mutation frequencies observed across five SARS-CoV-2 lineages, encompassing a total of 191,878 sequences. Individual DRN metrics displayed only modest power to predict the mutation frequency of individual residues. However, integrating the eight DRN metrics with additional structural and sequence-derived metrics allowed us to develop machine learning models which significantly improved the prediction of residue mutation frequency. While further refinements should enhance accuracy, we demonstrated a robust method to understand pathogen evolution. This approach can also guide the development of long-lasting drugs by targeting functional residues located in and near active site, and allosteric sites, that are less prone to mutations.

Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

The stability of the Opi1p repressor for phospholipid biosynthetic gene expression in Saccharomyces cerevisiae is dependent on its interactions with Scs2p and Ino2p

The yeast Saccharomyces cerevisiae Opi1p negatively regulates phospholipid biosynthetic genes. Under derepressing conditions, Opi1p binds to the endoplasmic reticulum/nuclear membrane with the aid of the membrane protein Scs2p and phosphatidic acids under derepressing conditions. Under repressing conditions, it enters the nucleus to inhibit the positive transcription factors Ino2p and Ino4p. While the spatial regulation of Opi1p is understood, the regulation of its abundance remains unclear. We investigated the role of Scs2p and Ino2p in Opi1p stability by overexpressing these proteins in yeast cells. Opi1p was stable in the presence of Scs2p, but mutations in residues required for interaction with Scs2p caused Opi1p unstable. Even in the absence of Scs2p, Opi1p remained stable in the strain having a mutation to increase phosphatidic acid levels. Conversely, overproduction of Ino2p reduced Opi1p stability, whereas a mutant Ino2p that cannot interact with Opi1p did not. Additionally, Opi1p was stable in strains lacking Ino2p or with a mutated Ino2p-binding domain. These findings suggest that regulation, adding another layer to the regulation of phospholipid biosynthetic gene expression by Opi1p.

Intricate Evolution of Multifunctional Lipoxygenase in Red Algae.

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations.

The Role of Cystic Fibrosis Transmembrane Conductance Regulator in Skeletal Muscle Contractile Function

Purpose: Genetic mutations in cystic fibrosis (CF) result in CF transmembrane conductance regulator (CFTR) dysfunction. CFTR is expressed in human skeletal muscle; its effect on skeletal muscle abnormalities is unknown. The study objective is to investigate the role of CFTR in skeletal muscle contractile function. Methods: We conducted a prospective, cross-sectional study comparing 34 adults with minimal and 18 with residual function CFTR mutations, recruited from Toronto Adult CF Centre, St. Michael's Hospital, Unity Health Toronto. Quadriceps, biceps brachii, and handgrip strength was measured with dynamometers; leg muscle power with the stair climb power test. Quadriceps muscle contractility was determined by quadriceps muscle strength normalized to quadriceps muscle size, measured with ultrasound images. Multivariable regression was used for analysis. Results: People with residual function CFTR mutations had higher quadriceps muscle torque normalized to quadriceps layer thickness and to rectus femoris cross-sectional area by 27.5 Nm/cm [95% CI (2.2, 52.8) Nm/cm, P = .034] and 5.6 Nm/cm2 [95% CI (0.3, 10.9) Nm/cm2, P = .041], respectively, compared with those with minimal function CFTR mutations. There were no differences in quadriceps muscle torque (P = .58), leg muscle power (P = .47), biceps brachii muscle force (P = .14), or handgrip force (P = .12) between the 2 mutation groups. Conclusions: CFTR protein may play a role in muscle contractility, implying a limited capacity to exert muscle force per unit of muscle size in people with CF. This suggests that building a greater muscle mass through resistance exercises focusing on muscle hypertrophy in exercise prescription may improve muscle strength in people with CF.

5143 Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity

Abstract Disclosure: H. Iwasaki: None. H. Suwanai: None. K. Kanekura: None. N. Satoshi: None. F. Yakou: None. H. Sakai: None. K. Ishii: None. N. hara: None. R. Suzuki: None. Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that disulfide bonds play an important role in TPO activity. However, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in the formation of disulfide bonds and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO. Methods: Vector plasmid TPO mutants C655F and C825R in CH patients were transfected into HEK293 cells. TPO activity and protein expression levels were measured by Amplex red assay and western blotting. The same procedure was performed in the presence of proteasome inhibitor MG132. Subcellular localization was determined by immunocytochemistry and flow cytometry. The location of all disulfide bonds within TPO was predicted by in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared to the wild-type (P < 0.05). In the presence of a proteasome inhibitor MG132, the protein expression level of TPO was increased to the level comparable with that of the wild-type but its activity did not. The degree of subcellular distribution of TPO to the cell surface in the mutant was lower than that in the wild-type TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO, and all TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation and membrane insertion. Presentation: 6/2/2024

Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease

In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7–21-fold lower DNA binding affinity and a 33–300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1’s protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.

Altered dNTP pools accelerate tumor formation in mice.

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP poolchanges in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosinetriphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

Identification of a novel RHCE*Ce (829G > A) allele associated with absence of C and e antigens expression.

The Rh blood group antigens are encoded by the RHD and RHCE genes, which possess a remarkable degree of polymorphism owing to their high homologous structures. These variants of the RH genes can lead to absence or weak expression of antigens. Analysis of RHCE genotyping by Polymerase Chain Reaction (PCR-SSP) method specific to detect c.48G, c.48C, 109 bp insertion of IVS2, c.201A and c.307C and RhCE phenotyping, were conducted in 316 Chinese patients in previous study. One patient with discrepancy typing result was collected for further RhCE serologic typing using microcolumn gel method and tube method in saline using monoclonal antibodies. PacBio sequencing was performed for RHCE, RHD and RHAG complete sequence analysis. 3D molecular models of the protein with the wild-type and mutant residue were generated using the DynaMut web server. The effect of the mutation on the protein function was predicted by PolyPhen-2 software. One male patient of Chinese Han was detected with RHCE*C allele showed by PCR-SSP method but ccEE phenotype. Further PacBio sequencing identified one normal RHCE*cE allele and one RHCE*Ce allele carried a novel c.829G > A (p.Gly277Arg) variant, which the encoded amino acid located in the ninth transmembrane segment of RhCE protein. Crystallisation analysis of 3D molecular models revealed that the substitution at Arg277 leads to the formation of additional hydrogen bonds, including weak hydrogen bonds between multiple atoms. It also results in hydrophobic ion interactions between Arg277 and Ala244. This mutation is predicted to have a damaging effect on protein function. One novel RHCE*Ce allele with c.829G > A (p.Gly277Arg) variant was identified to resulting in the absence or weak expression of C and e antigens.

A Study on the Binding Mechanism and the Impact of Key Residue Mutations between SND1 and MTDH Peptide through Molecular Dynamics Simulations.

Metastasis of breast cancer is the main cause of death for patients with breast cancer. The interaction between metadherin (MTDH) and staphylococcal nuclease domain 1 (SND1) plays a pivotal role in promoting breast cancer development. However, the binding details between MTDH and SND1 remain unclear. In this study, we employed all-atom molecular dynamics simulations (MDs) and conducted binding energy calculations to investigate the binding details and the impact of key residue mutations on binding. The mutations in key residues have not significantly affected the overall stability of the structure and the fluctuation of residues near the binding site; they have exerted a substantial impact on the binding of SND1 and MTDH peptide. The electrostatic interactions and van der Waals interactions play an important role in the binding of SND1 and the MTDH peptide. The mutations in the key residues have a significant impact on electrostatic and van der Waals interactions, resulting in weakened binding. The energy contributions of key residues mainly come from the electrostatic energy and van der Waals interactions of the side chain. In addition, the key residues form an intricate and stable network of hydrogen bonds and salt-bridge interactions with the MTDH peptide. The mutations in key residues have directly disrupt the interactions formed between SND1 and MTDH peptide, consequently leading to changes in the binding mode of the MTDH peptide. These analyses unveil the detailed atomic-level interaction mechanism between SND1 and the MTDH peptide, providing a molecular foundation for the development of antibreast cancer drugs.

The influence of residue interaction on thermal stability of lipase based on dynamic graph embedding

Lipases with high thermal stability have greater application in industrial production. Focusing on wild-type lipase and their thermotolerant mutants, this study employs dynamic graph embedding to explore the change of spatiotemporal characteristics in residue-residue interactions, analyzing the relationship between residue mutations and the thermal stability of lipase. K-means clustering is used to analyze the synergistic relationships between residues, revealing that thermotolerant mutants achieve higher thermal stability by enhancing the synergistic interactions between secondary structures, resulting in a more balanced and tightly-knit internal structure. Critical residues are identified using anomaly node detection methods, showing that residues with spatiotemporal instability are mainly distributed on highly flexible loop regions. Specifically, mutations at Tyr89 and Asp132 restrict the motion range of the loops, making the lipase structure more stable. The study demonstrates that mutating highly flexible regions to increase structural rigidity can effectively enhance the thermal stability of lipases.