A preliminary study about the potential risks of the UV-weathered microplastic: The proteome-level changes in the brain in response to polystyrene derived weathered microplastics.

The growing use of plastic materials has resulted in a constant increase in the risk associated with microplastics (MPs). Ultra-violet (UV) light and wind break down modify MPs in the environment into smaller particles known as weathered MPs (WMPs) and these processes increase the risk of MP toxicity. The neurotoxicity of weathered polystyrene-MPs remains unclear. Therefore, it is important to understand the risks posed by WMPs. We evaluated the chemical changes of WMPs generated under laboratory-synchronized environmentally mimetic conditions and compared them with virgin MPs (VMPs). We found that WMP had a rough surface, slight yellow color, reduced molecular weight, and structural alteration compared with those of VMP. Next, 2 µg of ∼100 µm in size of WMP and VMP were orally administered once a day for one week to C57BL/6 male mice. Proteomic analysis revealed that the WMP group had significantly increased activation of immune and neurodegeneration-related pathways compared with that of the VMP group. Consistently, in in vitro experiments, the human brain-derived microglial cell line (HMC-3) also exhibited a more severe inflammatory response to WMP than to VMP. These results show that WMP is a more profound inflammatory factor than VMP. In summary, our findings demonstrate the toxicity of WMPs and provide theoretical insights into their potential risks to biological systems and even humans in the ecosystem.

Computational design of a thermolabile uracil-DNA glycosylase of Escherichia coli.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.

Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density.

Serum amyloid A (SAA) is an acute-phase protein produced primarily in the liver that plays a key role in both the initiation and maintenance of inflammation. Rapidly secreted SAA induces neutrophilia at inflammatory sites, initiating inflammation and inducing the secretion of various cytokines, including TNF-α, IL-6, and IL-17. IL-17 is expressed in several inflammatory cells, including innate immune cells such as γδT cells, ILC3 cells, and neutrophils. Increased IL-17 levels exacerbate various inflammatory diseases. Among other roles, IL-17 induces bone loss by increasing receptor activator of nuclear factor-κB ligand (RANKL) secretion, which stimulates osteoclast differentiation. Several studies have demonstrated that chronic inflammation induces bone loss, suggesting a role for SAA in bone health. To test this possibility, we observed an increase in IL-17-producing innate immune cells, neutrophils, and γδT cells in these mice. In 6-month-old animals, we detected increased osteoclast-related gene expression and IL-17 expression in bone lysates. We also observed an increase in neutrophils that secreted RANKL in the bone marrow of TG mice. Finally, we demonstrated decreased bone mineral density in these transgenic (TG) mice. Our results revealed that the TG mice have increased populations of IL-17-producing innate immune cells, γδT cells, and neutrophils in TG mice. We additionally detected increased RANKL and IL-17 expression in the bone marrow of 6-month-old TG mice. Furthermore, we confirmed significant increases in RANKL-expressing neutrophils in TG mice and decreased bone mineral density. Our results provide evidence that chronic inflammation induced by SAA1 causes bone loss via IL-17-secreting innate immune cells.

NOLC1 knockdown suppresses prostate cancer progressions by reducing AKT phosphorylation and ß-catenin accumulation.

Although several studies have focused on cancer diagnosis and therapy, prostate cancer (PC) remains an intractable disease. Androgen deprivation therapy (ADT), which is used to treat early stage PC can lead to the development of castration-resistant prostate cancer (CRPC), which is highly associated with androgen receptor (AR) mutations. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a chaperone that shuttles between the nucleus and the cytoplasm. Studies suggest that NOLC1 regulates PC progression; however, the underlying mechanisms remain unclear. Herein, we showed that NOLC1 knockdown suppresses PC cell proliferation by altering the signaling pathways and the expression of various proteins involved in DNA replication, amino acid metabolism, and RNA processing. Mechanistically, NOLC1 knockdown suppressed cell cycle progression by inhibiting AKT phosphorylation and ß-catenin accumulation. Finally, we showed that NOLC1 expression is higher in human PC than in human hyperplastic prostate tissues. Altogether, we demonstrated that NOLC1 knockdown suppresses the progression of both AR-positive and AR-negative PC cells by inducing changes in the expression of several genes leading to cell cycle arrest. Thus, NOLC1 might be a novel and promising therapeutic target for PC.

Conformational switch that induces GDP release from Gi.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, ß, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gßγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.

Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or ß-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

Sibe: a computation tool to apply protein sequence statistics to predict folding and design in silico.

BACKGROUND: Evolutionary information contained in the amino acid sequences of proteins specifies the biological function and fold, but exactly what information contained in the protein sequence drives both of these processes? Considerable progress has been made to answer this fundamental question, but it remains challenging to explore the potential space of cooperative interactions between amino acids. Statistical analysis plays a significant role in studying such interactions and its use has expanded in recent years to studies ranging from coevolution-guided rational protein design to protein folding in silico. RESULTS: Here we describe a computational tool named Sibe for use in studies of protein sequence, folding, and design using evolutionary coupling between amino acids as a driving factor. In this study, Sibe is used to identify positionally conserved couplings between pairwise amino acids and aid rational protein design. In this process, pairwise couplings are filtered according to the relative entropy computed from the positional conservations and grouped into several 'blocks', which could contribute to driving protein folding and design. A human ß2-adrenergic receptor (ß2AR) was used to demonstrate that those 'blocks' contribute the rational design for specifying functional residues. Sibe also provides folding modules based on both the positionally conserved couplings and well-established statistical potentials for simulating protein folding in silico and predicting tertiary structure. Our results show that statistically inferences of basic evolutionary principles, such as conservations and coupled-mutations, can be used to rapidly design a diverse set of proteins and study protein folding. CONCLUSIONS: The developed software Sibe provides a computational tool for systematical analysis from protein primary to its tertiary structure using the evolutionary couplings as a driving factor. Sibe, written in C++, accounts for compatibility with the 'big data' era in biological science, and it primarily focuses on protein sequence analysis, but it is also applicable to extend to other modeling and predictions of experimental measurements.

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange.

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar.

Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative ß turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.

Benchmarking all-atom simulations using hydrogen exchange.

Long-time molecular dynamics (MD) simulations are now able to fold small proteins reversibly to their native structures [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. These results indicate that modern force fields can reproduce the energy surface near the native structure. To test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability and H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2° structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations.

Roles of the gate loop in ß-arrestin-1 conformational dynamics and phosphorylated receptor interaction.

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using ß-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of ß-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.

Veratramine Inhibits the Cell Cycle Progression, Migration, and Invasion via ATM/ATR Pathway in Androgen-Independent Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer-related death among men. Treatment of PC becomes difficult after progression because PC that used to be androgen-dependent becomes androgen-independent prostate cancer (AIPC). Veratramine, an alkaloid extracted from the root of the Veratrum genus, has recently been reported to have anticancer effects that work against various cancers; however, its anticancer effects and the underlying mechanism of action in PC remain unknown. We investigated the anticancer effects of veratramine on AIPC using PC3 and DU145 cell lines, as well as a xenograft mouse model. The antitumor effects of veratramine were evaluated using the CCK-8, anchorage-independent colony formation, trans-well, wound healing assays, and flow cytometry in AIPC cell lines. Microarray and proteomics analyses were performed to investigate the differentially expressed genes and proteins induced by veratramine in AIPC cells. A xenograft mouse model was used to confirm the therapeutic response and in vivo efficacy of veratramine. Veratramine dose dependently reduced the proliferation of cancer cells both in vitro and in vivo. Moreover, veratramine treatment effectively suppressed the migration and invasion of PC cells. The immunoblot analysis revealed that veratramine significantly downregulated Cdk4/6 and cyclin D1 via the ATM/ATR and Akt pathways, both of which induce a DNA damage response that eventually leads to G1 phase arrest. In this study, we discovered that veratramine exerted antitumor effects on AIPC cells. We demonstrated that veratramine significantly inhibited the proliferation of cancer cells via G0/G1 phase arrest induced by the ATM/ATR and Akt pathways. These results suggest that veratramine is a promising natural therapeutic agent for AIPC.

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC.

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

PACSY, a relational database management system for protein structure and chemical shift analysis.

PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu.

Unveiling OASIS family as a key player in hypoxia-ischemia cases induced by cocaine using generative adversarial networks.

Repeated cocaine use poses many serious health risks to users. One of the risks is hypoxia and ischemia (HI). To restore the biological system against HI, complex biological mechanisms operate at the gene level. Despite the complexity of biological mechanisms, there are common denominator genes that play pivotal roles in various defense systems. Among these genes, the cAMP response element-binding (Creb) protein contributes not only to various aspects of drug-seeking behavior and drug reward, but also to protective mechanisms. However, it is still unclear which Creb members are key players in the protection of cocaine-induced HI conditions. Herein, using one of the state-of-the-art deep learning methods, the generative adversarial network, we revealed that the OASIS family, one of the Creb family, is a key player in various defense mechanisms such as angiogenesis and unfolded protein response against the HI state by unveiling hidden mRNA expression profiles. Furthermore, we identified mysterious kinases in the OASIS family and are able to explain why the prefrontal cortex and hippocampus are vulnerable to HI at the genetic level.

Molecular basis of the PIP2-dependent regulation of CaV2.2 channel and its modulation by CaV ß subunits.

High-voltage-activated Ca2+ (CaV) channels that adjust Ca2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in an auxiliary CaV ß subunit-dependent manner. However, the molecular mechanism by which the ß subunits control the PIP2 sensitivity of CaV channels remains unclear. By engineering various α1B and ß constructs in tsA-201 cells, we reported that at least two PIP2-binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4II domain, exist in the CaV2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled CaV ß2 subunits. The membrane-anchored ß subunit abolished the PIP2 interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP2 sensitivity of CaV2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4II domain of CaV2.2 channels regardless of ß2 isotype. Our data demonstrated that the anchoring properties of CaV ß2 subunits to the plasma membrane determine the biophysical states of CaV2.2 channels by regulating PIP2 coupling to the nonspecific phospholipid-binding site in the I-II loop.

Dual Effects of Presynaptic Membrane Mimetics on α-Synuclein Amyloid Aggregation.

Aggregation of intrinsically disordered α-synuclein (αSN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of αSN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of αSN mutants under two types of liposomes, 1,2-Dioleoyl-sn-glycero-3-Phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of αSNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of αSNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of αSN, which is rationalized by the amyloidogenic structure of αSN and condensation-dilution of local αSN concentration. Finally, we propose physicochemical properties of αSN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

Structural mechanism for regulation of Rab7 by site-specific monoubiquitination.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

Uncovering symmetry-breaking vector and reliability order for assigning secondary structures of proteins from atomic NMR chemical shifts in amino acids.

Unravelling the complex correlation between chemical shifts of (13) C (α), (13) C (ß), (13) C', (1) H (α), (15) N, (1) H ( N ) atoms in amino acids of proteins from NMR experiment and local structural environments of amino acids facilitates the assignment of secondary structures of proteins. This is an important impetus for both determining the three-dimensional structure and understanding the biological function of proteins. The previous empirical correlation scores which relate chemical shifts of (13) C (α), (13) C (ß), (13) C', (1) H (α), (15) N, (1) H ( N ) atoms to secondary structures resulted in progresses toward assigning secondary structures of proteins. However, the physical-mathematical framework for these was elusive partly due to both the limited and orthogonal exploration of higher-dimensional chemical shifts of hetero-nucleus and the lack of physical-mathematical understanding underlying those correlation scores. Here we present a simple multi-dimensional hetero-nuclear chemical shift score function (MDHN-CSSF) which captures systematically the salient feature of such complex correlations without any references to a random coil state of proteins. We uncover the symmetry-breaking vector and its reliability order not only for distinguishing different secondary structures of proteins but also for capturing the delicate sensitivity interplayed among chemical shifts of (13) C (α), (13) C (ß), (13) C', (1) H (α), (15) N, (1) H ( N ) atoms simultaneously, which then provides a straightforward framework toward assigning secondary structures of proteins. MDHN-CSSF could correctly assign secondary structures of training (validating) proteins with the favourable (comparable) Q3 scores in comparison with those from the previous correlation scores. MDHN-CSSF provides a simple and robust strategy for the systematic assignment of secondary structures of proteins and would facilitate the de novo determination of three-dimensional structures of proteins.

Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues.

Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz-Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding-unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined.