Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Redox Regulation and Metabolic Dependency of Zika Virus Replication: Inhibition by Nrf2-Antioxidant Response and NAD(H) Antimetabolites.

Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Hierarchal polyaniline-folic acid nanostructures act as a platform for electrochemical detection of tumor cells.

The fabrication of electrochemical sensing platforms for cancer monitoring by quantifying circulating tumor cells (CTCs) in blood holds promise for providing a low-cost, rapid, feasible, and safe approach for cancer diagnosis. Here, we isolate cancer cells using CoFe2O4 nanoparticles functionalized with folic acid and chitosan as an inexpensive magnetic nanoprobe. This electrochemical cytosensing platform was realized using polyaniline-folic acid nanohybrids with a three-dimensional hierarchical structure that presents abundant affinity sites toward overexpressed folate bioreceptors on cancer cells, in addition to retaining satisfied conductivity. Furthermore, 3D modeling and simulation of the polyaniline-folic acid structures were conducted to investigate the stable complex between aniline and folate, and the interaction between the polyaniline-folate complex and folate receptor alpha1, a bioreceptor on MCF-7 was revealed for the first time. The limit of detection was calculated to be 4 cells mL-1 with a linear range from 50 to 106 cells mL-1.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

Mechanistic Target of Rapamycin Signaling Activation Antagonizes Autophagy To Facilitate Zika Virus Replication.

Zika virus (ZIKV), a mosquito-transmitted flavivirus, is linked to microcephaly and other neurological defects in neonates and Guillain-Barré syndrome in adults. The molecular mechanisms regulating ZIKV infection and pathogenic outcomes are incompletely understood. Signaling by the mechanistic (mammalian) target of rapamycin (mTOR) kinase is important for cell survival and proliferation, and viruses are known to hijack this pathway for their replication. Here, we show that in human neuronal precursors and glial cells in culture, ZIKV infection activates both mTOR complex 1 (mTORC1) and mTORC2. Inhibition of mTOR kinase by Torin1 or rapamycin results in reduction in ZIKV protein expression and progeny production. Depletion of Raptor, the defining subunit of mTORC1, by small interfering RNA (siRNA) negatively affects ZIKV protein expression and viral replication. Although depletion of Rictor, the unique subunit of mTORC2, or the mTOR kinase itself also inhibits the viral processes, the extent of inhibition is less pronounced. Autophagy is transiently induced early by ZIKV infection, and impairment of autophagosome elongation by the class III phosphatidylinositol 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA) enhances viral protein accumulation and progeny production. mTOR phosphorylates and inactivates ULK1 (S757) at later stages of ZIKV infection, suggesting a link between autophagy inhibition and mTOR activation by ZIKV. Accordingly, inhibition of ULK1 (by MRT68921) or autophagy (by 3-MA) reversed the effects of mTOR inhibition, leading to increased levels of ZIKV protein expression and progeny production. Our results demonstrate that ZIKV replication requires the activation of both mTORC1 and mTORC2, which negatively regulates autophagy to facilitate ZIKV replication.IMPORTANCE The re-emergence of Zika virus (ZIKV) and its association with neurological complications necessitates studies on the molecular mechanisms that regulate ZIKV pathogenesis. The mTOR signaling cascade is tightly regulated and central to normal neuronal development and survival. Disruption of mTOR signaling can result in neurological abnormalities. In the studies reported here, we demonstrate for the first time that ZIKV infection results in activation of both mTORC1 and mTORC2 to promote virus replication. Although autophagy is activated early in infection to counter virus replication, it is subsequently suppressed by mTOR. These results reveal critical roles of mTOR signaling and autophagy in ZIKV infection and point to a possible mechanism underlying ZIKV-induced pathogenesis. Elucidating the role of mTOR signaling in ZIKV infection will provide insights into the mechanisms of ZIKV-induced neurological complications and potential targets for therapeutic approaches.

High-Throughput Screening at the Membrane Interface Reveals Inhibitors of Amyloid-ß.

Aggregation and the formation of oligomeric intermediates of amyloid-ß (Aß) at the membrane interface of neuronal cells are implicated in the cellular toxicity and pathology of Alzheimer's disease. Small molecule compounds have been shown to suppress amyloid aggregation and cellular toxicity, but often the presence of a lipid membrane negates their activity. A high-throughput screen of 1800 small molecules was performed to search for membrane active inhibitors, and 21 primary hits were discovered. Through the use of fluorescence-based assays, transmission electron microscopy, and dot blot assays, the initial 21 primary hits were narrowed down to five lead compounds. Nuclear magnetic resonance and circular dichroism experiments were used for further confirmation of amyloid inhibition at the membrane interface and to obtain insights into the secondary structure of amyloid-ß, while size exclusion chromatography was used to characterize the size of Aß species. Lastly, dye-leakage assays allowed us to understand how the addition of the five lead compounds affected amyloid-ß's ability to permeate the lipid bilayer. These results provide insights into small molecules that stabilize small amyloid species in the presence of membranes for the development of tool compounds for deeper investigations of these transient species.

Lifespan associated global patterns of coherent neural communication.

Healthy ageing is accompanied by changes to spontaneous electromagnetic oscillations. At the macroscopic scale, previous studies have quantified the basic features, e.g., power and frequencies in rhythms of interest from the perspective of attention, perception, learning and memory. On the other hand, signatures and modes of neural communication have recently been argued to be identifiable from global measures applied on neuro-electromagnetic data such as global coherence that quantifies the degree of togetherness of distributed neural oscillations and metastability that parametrizes the transient dynamics of the network switching between successive stable states. Here, we demonstrate that global coherence and metastability can be informative measures to track healthy ageing dynamics over lifespan and together with the traditional spectral measures provides an attractive explanation of neuronal information processing. Finding normative patterns of brain rhythms in resting state MEG would naturally pave the way for tracking task relevant metrics that could crucially determine cognitive flexibility and performance. While previously reported observations of a reduction in peak alpha frequency and increased beta power in older adults are reflective of changes at individual sensors (during rest and task), global coherence and metastability pinpoint the underlying coordination dynamics over multiple brain areas across the entire lifespan. In addition to replication of the previous observations in a substantially larger lifespan cohort than what was previously reported, we also demonstrate, for the first time to the best of our knowledge, age related changes in coherence and metastability in signals over time scales of neuronal processing. Furthermore, we observed a marked frequency dependence in changes in global coordination dynamics, which, coupled with the long-held view of specific frequency bands subserving different aspects of cognition, hints at differential functional processing roles for slower and faster brain dynamics.

High-Speed Atomic Force Microscopy Reveals the Structural Dynamics of the Amyloid-ß and Amylin Aggregation Pathways.

Individual Alzheimer's disease (AD) patients have been shown to have structurally distinct amyloid-ß (Aß) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aß fibril structures. Thus, the characterization of the structural dynamics of Aß could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aß and amylin, which is associated with type II diabetes, shares some common biological features with Aß, and is reported to be involved in AD.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Spontaneous Lipid Nanodisc Fomation by Amphiphilic Polymethacrylate Copolymers.

There is a growing interest in the use of lipid bilayer nanodiscs for various biochemical and biomedical applications. Among the different types of nanodiscs, the unique features of synthetic polymer-based nanodiscs have attracted additional interest. A styrene-maleic acid (SMA) copolymer demonstrated to form lipid nanodiscs has been used for structural biology related studies on membrane proteins. However, the application of SMA polymer based lipid nanodiscs is limited because of the strong absorption of the aromatic group interfering with various experimental measurements. Thus, there is considerable interest in the development of other molecular frameworks for the formation of polymer-based lipid nanodiscs. In this study, we report the first synthesis and characterization of a library of polymethacrylate random copolymers as alternatives to SMA polymer. In addition, we experimentally demonstrate the ability of these polymers to form lipid bilayer nanodiscs through the fragmentation of lipid vesicles by means of light scattering, electron microscopy, differential scanning calorimetry, and solution and solid-state NMR experiments. We further demonstrate a unique application of the newly developed polymer for kinetics and structural characterization of the aggregation of human islet amyloid polypeptide (also known as amylin) within the lipid bilayer of the polymer nanodiscs using thioflavin-T-based fluorescence and circular dichroism experiments. Our results demonstrate that the reported new styrene-free polymers can be used in high-throughput biophysical experiments. Therefore, we expect that the new polymer nanodiscs will be valuable in the structural studies of amyloid proteins and membrane proteins by various biophysical techniques.

Understanding the distinguishable structural and functional features in zebrafish TLR3 and TLR22, and their binding modes with fish dsRNA viruses: an exploratory structural model analysis.

Viral infections are one of the major challenges in aquaculture production, and considered as the potential threat for fish farming. Toll-like receptor (TLR) 3 and TLR22 are highly specialized innate immune receptors that recognize double-stranded (ds)-RNA of viruses resulting in the induction of innate immunity. The existence of TLR3 and TLR22 only in aquatic animals indicates their distinctive characteristics in viral infection; however, the studies in exploring their structural features and dsRNA binding mechanism are still elusive. Here, we studied the structural and functional differentiations of TLR3 and TLR22 in zebrafish by employing comparative modeling and molecular dynamics simulation. Comparative structural analysis revealed a distinct spatial arrangement of TLR22 ectodomain with a flattened horseshoe-shape conformation as compared to other TLRs. Essential dynamics studies showed that unlike TLR3, TLR22 possessed a prominent motion, elasticity and twisting at both terminus separated by a distance equivalent to the length of a short-sized dsRNA. Interaction analysis of polyinosinic:polycytidylic acid (poly I:C) and dsRNA depicted leucine-rich-repeats (LRR)2-3 and LRR18-19 (in TLR3) and LRRNT-LRR3 and LRR22-24 (in TLR22) as the potential binding sites. The short-sized dsRNA binds tightly across its full-length with TLR22-monomer, and suggested that TLR22 dimer may sense long-sized dsRNA. Binding energy (BE) calculation using MM/PBSA method from the TLR3- and TLR22-ligand complexes revealed an adequate binding affinity between TLR22-monomer and dsRNA as like as TLR3-dimer-dsRNA complex. Mutagenesis and BE computation of key residues suggested their involvement in dsRNA recognition. These findings can be helpful for therapeutic applications against viral diseases in fish.

Structural insights into the MDP binding and CARD-CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach.

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)-a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2-leucine rich repeat (LRR) domain activates NF-κB signaling through a protein-protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD-CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD-CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2-LRR confer MDP recognition by hydrophobic and hydrogen bond (H-bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2-CARDa and the electronegative surface on zRIP2-CARD reinforced mostly by H-bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2-CARDa and Asp494 of zRIP2-CARD. Arg11 and Lys53 of zNOD2-CARDa and Ser498 and Glu508 of zRIP2-CARD are critical residues for CARD-CARD interaction and NOD2 signaling. The 2.7 Å H-bond between Lys104 of the linker and Glu508 of zRIP2-CARD suggests a possible role of the linker for shaping CARD-CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD-CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective.

Backbone 1H, 13C, and 15N chemical shift assignments for human SERF2.

Human small EDRK-rich factor protein SERF2 is a cellular driver of protein amyloid formation, a process that has been linked to neurodegenerative diseases including Alzheimer's and Parkinson's disease. SERF2 is a 59 amino acid protein, highly charged, and well conserved whose structure and physiological function is unclear. SERF family proteins including human SERF2 have shown a tendency to form fuzzy complexes with misfolded proteins such as α-Synuclein which has been linked to Parkinson's disease. SERF family proteins have been recently identified to bind nucleic acids, but the binding mechanism(s) remain enigmatic. Here, using multidimensional solution NMR, we report the 1H, 15N, and 13C chemical shift assignments (~ 86% of backbone resonance assignments) for human SERF2. TALOS-N predicted secondary structure of SERF2 showed three very short helices (3-4 residues long) in the N-terminal region of the protein and a long helix in the C-terminal region spanning residues 37-46 which is consistent with the helical content indicated by circular dichroism spectroscopy. Paramagnetic relaxation enhancement NMR analysis revealed that a short C-terminal region E53-K55 is in the proximity of the N-terminus. Having the backbone assignment of SERF2 allowed us to probe its interaction with α-Synuclein and to identify the residues in SERF2 binding interfaces that likely promote α-Synuclein aggregation.

Protein Nanoparticles as Vaccine Platforms for Human and Zoonotic Viruses.

Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.

Amyloid Accelerator Polyphosphate Implicated as the Mystery Density in α-Synuclein Fibrils.

Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the two critical lysine residues K43 and K45 leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.

Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

Direct interaction between the transmembrane helices stabilize cytochrome P450 2B4 and cytochrome b5 redox complex.

The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.