Homodimeric Protein Research Articles

Transforming growth factor-β signaling in head and neck squamous cell carcinoma: Insights into cellular responses.

Head and neck squamous cell carcinoma (HNSCC) arises in the oral cavity, salivary glands, larynx, pharynx, nasal cavity and paranasal sinuses, and is characterized by high morbidity and metastasis rates. Transforming growth factor-β (TGF-β) is a homodimeric protein known to be a multifunctional regulator in target cells and to serve a pivotal role in numerous types of cancer, including HNSCC. The role of TGF-β signaling in carcinogenesis can change from tumor-suppressing to tumor-promoting. In addition, TGF-β induces epithelial-mesenchymal transition and restrains immune surveillance on malignant cells. In the present review, the effects of TGF-β signaling at a cellular level were discussed, which includes the regulation of tumor cells, immune cells and other stromal cells, as well as the possible mechanisms underlying the conversion from a tumor suppressor to a tumor promoter in HNSCC. Further research is required to improve the understanding on how this network is involved in carcinogenesis, progression and metastases in HNSCC.

Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu2+ binding properties of Calgranulin C. We demonstrate that the two Cu2+ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu2+-bound protein clearly show two distinct components at higher Cu2+:protein ratios, which is indicative of the two different binding environments for the Cu2+ ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca2+-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu2+ ions. To further probe the Cu2+ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu2+ binding site dominates the other. At both sites we see distinct signatures of Cu2+–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu2+ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu2+ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu2+–Cu2+ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu2+–Cu2+ distance when oligomerization occurs.

Structural basis for promotion of duodenal iron absorption by enteric ferric reductase with ascorbate

Dietary iron absorption is regulated by duodenal cytochrome b (Dcytb), an integral membrane protein that catalyzes reduction of nonheme Fe3+ by electron transfer from ascorbate across the membrane. This step is essential to enable iron uptake by the divalent metal transporter. Here we report the crystallographic structures of human Dcytb and its complex with ascorbate and Zn2+. Each monomer of the homodimeric protein possesses cytoplasmic and apical heme groups, as well as cytoplasmic and apical ascorbate-binding sites located adjacent to each heme. Zn2+ coordinates to two hydroxyl groups of the apical ascorbate and to a histidine residue. Biochemical analysis indicates that Fe3+ competes with Zn2+ for this binding site. These results provide a structural basis for the mechanism by which Fe3+ uptake is promoted by reducing agents and should facilitate structure-based development of improved agents for absorption of orally administered iron.

Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography-Capillary Zone Electrophoresis-Tandem Mass Spectrometry.

Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size-exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for the characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics.

Processivity of dimeric kinesin-1 molecular motors.

Kinesin‐1 is a homodimeric motor protein that can move along microtubule filaments by hydrolyzing ATP with a high processivity. How the two motor domains are coordinated to achieve such high processivity is not clear. To address this issue, we computationally studied the run length of the dimer with our proposed model. The computational data quantitatively reproduced the puzzling experimental data, including the dramatically asymmetric character of the run length with respect to the direction of external load acting on the coiled‐coil stalk, the enhancement of the run length by addition of phosphate, and the contrary features of the run length for different types of kinesin‐1 with extensions of their neck linkers compared with those without extension of the neck linker. The computational data on other aspects of the movement dynamics such as velocity and durations of one‐head‐bound and two‐head‐bound states in a mechanochemical coupling cycle were also in quantitative agreement with the available experimental data. Moreover, predicted results are provided on dependence of the run length upon external load acting on one head of the dimer, which can be easily tested in the future using single‐molecule optical trapping assays.

PO-096 Natural variation in serum small non-coding RNAs – potential biomarkers of cancer

IntroductionIn recent years, immunotherapy including checkpoint blockade acts as potential strategies to cure cancer. A number of recent studies have identified inducible co-stimulator (ICOS) as a crucial player in the immune checkpoints blockade therapy. ICOS, a homodimeric protein which belongs to CD28 superfamily, is expressed on activated T cells and resting memory T cells. Upon inflammation, ICOS and its ligand ICOSL (B7RP-1, B7H-2) have been shown to affect T cell responses via ICOS-mediated PI3-kinase signalling to enhance the T cells development and activity. In addition, previous studies have shown that upon CTLA-4 blockade, the ICOS/ICOSL pathway plays an important role in tumour immunity.In this study, we aim to develop and implement the Fc-fusion recombinant ICOSL proteins to induce T cells activation for therapeutic use. Our data suggested that recombinant ICOSL-Fc can enhance the proliferation and cytotoxicity effect of human Pan T cells by increasing INFg and IL10 secretion. Moreover, ICOSL-Fc can also inhibit tumour growth in xenograft animal models. Based on our study, ICOS/ICOSL pathway could be a promising therapeutic target for the immunotherapy.Material and methodsExpression and Purification of Recombinant ICOSL ProteinsConstruction of Fc-fusion ICOSL proteins:ICOS-mFc,mICOS-mFc, andICOSL-FcExpression and purification of recombinant ICOSL proteinsEmploy Flow Cytometry and ELISA-Based Assay to Confirm ICOSL-Fc Fusion Protein Can Bind to ICOSPerform ICOS binding ELISA assay with fusion ICOSL-Fc protein.Perform Pan T cell flow binding assay with fusion ICOSL-Fc protein.Results and discussionsICOSL-Fc can induce secretion of INFγ and IL-10, and proliferation of human Pan T cells.The tumour specific ICOSL-Fc engagement increases the cytotoxicity of human PBMCConclusionExpressions of PD1, ICOS and CTLA4 can be observed in PBMC under CD3/CD28 stimulation on day 1, day 3, and day 5.Recombinant human ICOSL-Fc protein was able to bind to both human ICOS and mouse ICOS ECD domains.Cell-base immunofluorescence was employed to illustrate recombinant human ICOSL-Fc protein was able to bind activated T cells.3H-Thymidine incorporation study illustrated that recombinant ICOSL-Fc was able to increase T cells proliferation.In vitro efficacy assay suggested that recombinant ICOSL-Fc can remarkable increase IL-10 and INFγ secretion on human pan T cells.The preliminary T cell cytotoxicity assay showed ICOSL-Fc treatment can increase the killing effect.

Abstract CT126: A phase I first time in human study to evaluate the safety, pharmacokinetics, and immunogenicity of MEDI5083 alone and in combination with durvalumab in selected advanced solid tumors

Abstract Background CD40 is a member of the TNFR superfamily that is broadly expressed by immune, vascular, epithelial, and tumor cells. Interaction between CD40 and its ligand CD40L plays an important role in innate and adaptive immunity. APCs such as dendritic cells can be primed by CD4+ T cells by interaction between CD40 and CD40L. Several studies have confirmed that CD40 is broadly expressed on multiple solid tumor types. Thus, further evaluation of investigational agents and combination immunotherapies targeting the CD40 pathway is warranted across multiple cancer indications. MEDI5083 is a homodimeric fusion protein consisting of 3 scCD40L domains and an IgG4P Fc domain. MEDI5083 agonizes CD40 on APCs and bridges the innate and adaptive immune systems by promoting potent antitumor T cell mediated immune responses. Durvalumab is a selective, high-affinity, engineered human IgG1 mAb that blocks PD-L1 binding to PD-1 and CD80, allowing T cells to recognize and kill tumor cells. In clinical studies durvalumab has shown a manageable and consistent tolerability profile across multiple tumor types. The combination of MEDI5083 and durvalumab has the potential to activate the adaptive immune system through potentially complementary mechanisms, leading to enhanced targeting of tumor cells. Methods This Phase 1 first in human study evaluates the safety, pharmacokinetics, and immunogenicity of MEDI5083 alone and in combination with durvalumab in select advanced solid tumors. The primary objective is to evaluate the safety and tolerability, describe dose-limiting toxicity, and determine the maximum tolerated dose or the highest protocol-defined dose for MEDI5083 when administered as monotherapy and when administered concurrently in combination with durvalumab. Key secondary objectives include describing PK, systemic pharmacodynamics, immunogenicity and preliminary clinical activity of MEDI5083 administered alone or concurrently with durvalumab. This is a 3-part study; Part 1 is dose escalation of MEDI5083 monotherapy followed by either durvalumab monotherapy or potential reinduction with MEDI5083. Part 2 is dose escalation of MEDI5083 with concurrent durvalumab therapy, followed by durvalumab monotherapy restricted to selected tumor types. Part 3 is MEDI5083 with concurrent durvalumab, followed by durvalumab monotherapy in tumor specific expansion cohorts. Recruitment is ongoing (NCT03089645). Citation Format: Ben Tran, Johanna Bendell, Martin Gutierrez, Jie Yang, Natasha Angra, Victoria L. Chiou, Mark Voskoboynik. A phase I first time in human study to evaluate the safety, pharmacokinetics, and immunogenicity of MEDI5083 alone and in combination with durvalumab in selected advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT126.

Abstract 5760: An IgG1 version of the anti-TfR1 antibody ch128.1 shows significant antitumor activity against different xenograft models of multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also identified as CD71, is a type II transmembrane homodimeric protein involved in the cellular uptake of iron and in the regulation of cell growth. TfR1 is a meaningful target for antibody-based cancer immunotherapy due to its high expression levels on the surface of cancer cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits antitumor activity in SCID-Beige mice bearing disseminated multiple myeloma (MM) KMS-11 cells. Since human IgG1 has been the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared to IgG3, including a more established developability, we constructed an IgG1 version of ch128.1. This novel antibody, ch128.1/IgG1, expressed in murine cells (NS0/1), is properly assembled and secreted and binds antigen (TfR1) similar to its IgG3 counterpart, as demonstrated by ELISA and flow cytometry. Administration of a single dose of the IgG1 completely blocks subcutaneous KMS-11 tumor formation in SCID-Beige mice. Importantly, a single dose of the antibody also shows significant protection, including long-term survival, in this same strain of mice bearing disseminated human MM cells KMS-11 (Asian origin) or MM.1S (African American origin), at different stages of the disease. As expected, the protection was stronger when the mice were treated at an earlier disease stage. Studies using MM.1S cells are of particular relevance given the higher incidence and mortality of MM in African Americans compared to other racial groups. Importantly, we also found that ch128.1/IgG1 confers in vivo protection against MM.1R cells, the MM.1S variant that is dexamethasone resistant. We also developed a ch128.1/IgG1 triple mutant (L234A/L235A/P329S) antibody that does not bind to FcγRs and C1q (the initial protein of the complement pathway), but retains TfR1 binding, as well as binding to FcRn, the neonatal Fc receptor (also known as the Brambell receptor), which is critical in retaining antibody bioavailability in the blood. The triple mutant failed to confer protection in vivo against KMS-11 and MM.1S cells, suggesting a critical role of the antibody Fc fragment in ch128.1/IgG1-mediated antitumor activity, consistent with a potential role of functional effector cells present in this mouse model, such as macrophages. In fact, we found that ch128.1/IgG1 elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in the presence of murine bone marrow derived macrophages as effectors and KMS-11 cells as targets, similar to its IgG3 counterpart. Taken together, our results suggest that ch128.1/IgG1 has great potential as an effective therapy for incurable human B-cell malignancies such as MM. Citation Format: Tracy R. Daniels-Wells, Pierre V. Candelaria, Lai Sum Leoh, Otoniel Martinez-Maza, Manuel L. Penichet. An IgG1 version of the anti-TfR1 antibody ch128.1 shows significant antitumor activity against different xenograft models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5760.

An Assembly-Activating Site in the Hepatitis B Virus Capsid Protein Can Also Trigger Disassembly.

The Hepatitis B Virus (HBV) core protein homodimers self-assemble to form an icosahedral capsid that packages the viral genome. Disassembly occurs in the nuclear basket to release the mature genome to the nucleus. Small molecules have been developed that bind to a pocket at the interdimer interface to accelerate assembly and strengthen interactions between subunits; these are under development as antiviral agents. Here, we explore the role of the dimer-dimer interface by mutating sites in the drug-binding pocket to cysteine and examining the effect of covalently linking small molecules to them. We find that ligands bound to the pocket may trigger capsid disassembly in a dose-dependent manner. This result indicates that, at least transiently, the pocket adopts a destabilizing conformation. We speculate that this pocket also plays a role in virus disassembly and genome release by binding ligands that are incompatible with virus stability, "unwanted guests." Investigating protein-protein interactions, especially large protein polymers, offers new and unique challenges. By using an engineered addressable thiol, we provide a means to examine the effects of modifying an interface without requiring drug-like properties for the ligand.

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA–protein and protein–protein interactions.

Interplay of self-association and conformational flexibility in regulating protein function.

Many functional roles have been attributed to homodimers, the most common mode of protein self-association, notably in the regulation of enzymes, ion channels, transporters and transcription factors. Here we review findings that offer new insights into the different roles conformational flexibility plays in regulating homodimer function. Intertwined homodimers of two-domain proteins and their related family members display significant conformational flexibility, which translates into concerted motion between structural domains. This flexibility enables the corresponding proteins to regulate function across family members by modulating the spatial positions of key recognition surfaces of individual domains, to either maintain subunit interfaces, alter them or break them altogether, leading to a variety of functional consequences. Many proteins may exist as monomers but carry out their biological function as homodimers or higher-order oligomers. We present early evidence that in such systems homodimer formation primes the protein for its functional role. It does so by inducing elevated mobility in protein regions corresponding to the binding epitopes of functionally important ligands. In some systems this process acts as an allosteric response elicited by the self-association reaction itself. Our analysis furthermore suggests that the induced extra mobility likely facilitates ligand binding through the mechanism of conformational selection.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Small molecule inhibitors of mammalian thioredoxin reductase as potential anticancer agents: An update.

Mammalian thioredoxin reductase (TrxR) enzymes are homodimeric flavin proteins sharing a unique yet essential selenocysteine residue at their C-terminus. TrxRs, together with their endogenous substrate thioredoxins, play a crucial role in regulating diverse cellular redox events. A wealth of evidence from both clinic observations and bench studies supports that overactivation/dysfunction of TrxRs has a close link to the onset and development of various diseases, such as cancer and neurodegeneration. Thus, an increasing interest has been attracted to find small molecule modulators of TrxRs during the past years. Herein, we briefly discussed the relevance of targeting TrxRs inhibition for cancer treatment, and presented the small molecule inhibitors of mammalian TrxRs published in the nonpatent literatures from 2011 to 2016. The mechanisms of inhibition by different classes of molecules were summarized, and some inhibitors with promising anticancer activity were further discussed. We expect this work would be a comprehensive reference in the medicinal chemistry, and have a broad audience across multiple disciplines.

Small Gd(III) Tags for Gd(III)-Gd(III) Distance Measurements in Proteins by EPR Spectroscopy.

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd ( S configuration) and C8-Gd ( R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band 1H-ENDOR (electron-electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron-electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.

Asymmetric Configurations in a Reengineered Homodimer Reveal Multiple Subunit Communication Pathways in Protein Allostery

Many allosteric proteins form homo‐oligomeric complexes to regulate a biological function. In homo‐oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo‐oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand‐induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered, single‐chain version of the homodimeric transcription factor cAMP Receptor Protein (CRPSC). By combining wild‐type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit remains wild‐type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild‐type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo‐oligomers employ to transduce signals.Support or Funding InformationThis work was supported with startup funds from Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY.

Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.

Backbone and Side-Chain (1)H, (13)C, and (15)N Chemical Shift Assignments for the homodimeric Histone-like DNA binding protein (Hup) of Helicobacter pylori

Backbone and Side-Chain (1)H, (13)C, and (15)N Chemical Shift Assignments for the homodimeric Histone-like DNA binding protein (Hup) of Helicobacter pylori

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins.

Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins (‘Transcription Activator-Like Effector Dimers’; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

Native prion protein homodimers are destabilized by oligomeric amyloid β 1–42 species as shown by single-molecule imaging

Prion proteins (PrPc) are receptors for amyloid β 1-42 (Aβ1-42) oligomers, but we do not know the impact of Aβ1-42 binding to PrPc on the interaction of membrane-bound PrPc with molecules that regulate downstream biological pathways. Stability of the PrPc dimeric complex and subsequent intermolecular interactions with membranous or cytoplasmic molecules are important for physiological functions of PrPc including neuroprotection. The principal aim of this study was to determine whether homodimer lifetime of PrPc is affected by the presence of Aβ1-42 oligomers. Single-molecule imaging analysis was carried out by total internal reflection fluorescence microscopy in PrPc-transfected CHO-K1 cells in the absence or presence of characterized Aβ1-42 oligomers. The contribution of different Aβ1-42 oligomer conformations to Alzheimer's disease pathophysiology and to the associated neurotoxicity is unknown. To be precise, with the oligomeric species used in our study, we biochemically analyzed the molecular weight of oligomers formed from Aβ1-42 monomers under our experimental conditions. The lifetime of PrPc homodimers was 210 ms, and in the presence of Aβ1-42 oligomers, the lifetime was significantly reduced (to 92 ms). The reduction of PrPc homodimer lifetime by Aβ1-42 oligomers may impair PrPc-mediated downstream neuroprotective signaling.

PDB-NMA of a protein homodimer reproduces distinct experimental motility asymmetry

We have extended our analytically derived PDB-NMA formulation, Atomic Torsional Modal Analysis or ATMAN (Tirion and ben-Avraham 2015 Phys. Rev. E 91 032712), to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3 resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50 sphere, reproduces the distinct experimental Debye–Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

Exploring the association of rs10490924 polymorphism with age-related macular degeneration: An in silico approach

The polymorphism rs10490924 (A69S) in the age-related maculopathy susceptibility 2 (ARMS2) gene is highly associated with age-related macular degeneration, which is the leading cause of blindness among the elderly population. ARMS2 gene encodes a putative small (11 kDa) protein, which the function and localization of the ARMS2 protein remain under debate. For a better understanding of functional impacts of A69S mutation, we performed a detailed analysis of an ARMS2 sequence with a broad set of bioinformatics tools. In silico analysis was followed to predict the tertiary structure, putative binding site regions, and binding site residues. Also, the effects of this mutation on protein stability, aggregation propensity, and homodimerization were analyzed. Next, a molecular dynamic simulation was carried out to understand the dynamic behavior of wild-type, A69S, and phosphorylated A69S structures. The results showed alterations in the putative post-translational modification sites on the ARMS2 protein, due to the mutation. Furthermore, the stability of protein and putative homodimer conformations were affected by the mutation. Molecular dynamic simulation results revealed that A69S mutation enhances the rigidity of the ARMS2 structure and residue serine at position 69 is buried and may not be phosphorylated; however, phosphorylated serine enhances the flexibility of the ARMS2 structure. In conclusion, our study provides new insights into the deleterious effects of A69S mutation on the ARMS2 structure.