Core Protein Complex Research Articles

Exploring the Active Ingredients and Mechanism of Action of Huanglian Huazhuo Capsule for the Treatment of Obese Type-2 Diabetes Mellitus Based on Using Network Pharmacology and Molecular Docking.

Background Obese type 2 diabetes mellitus (obese T2DM) is one of the prime diseases that endangers human health. Clinical studies have confirmed the ability of the Huanglian Huazhuo capsule to treat obese T2DM; however, its mechanism of action is still unclear. In this study, effects and mechanisms of the Huanglian Huazhuo capsule in obese T2DM were systematically investigated using network pharmacology and molecular docking techniques. Methods The active ingredients and targets of the Huanglian Huazhuo capsule were extracted from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Obese T2DM diabetes-related targets were retrieved from a geographic dataset combined with a gene card database. A protein-protein interaction (PPI) network was constructed to screen core targets. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using Database for Annotation Visualization and Integrated Discovery (DAVID). Interactions between potential targets and active compounds were assessed using molecular docking. Molecular docking was performed on the best core protein complexes obtained using molecular docking. Results A total of 89 and 108 active ingredients and targets, respectively, were identified. Seven core targets were obtained using a topological analysis of the PPI network. The GO and KEGG pathway enrichment analyses showed that the effects of the Huanglian Huazhuo capsules were mediated by inflammation, lipid response, oxidative stress-related genes, and HIF-1 and IL-17 signaling pathways. Good binding ability was observed between the active compounds and screened targets using molecular docking. Conclusions The active ingredients, potential targets, and pathways of the Huanglian Huazhuo capsule for the treatment of obese T2DM were successfully predicted, providing a new strategy for further investigation of its molecular mechanisms. In addition, the potential active ingredients provide a reliable source for drug screening in obese T2DM.

ROR2-Vangl2 complexes direct ovarian cancer cell motility in response to Wnt5a (265)

<b>Objectives:</b> In both developmental and cancer contexts, collective migration has shown to be at least partially dependent upon asymmetric localization of core protein complexes within leader and follower cells. A critical function of non-canonical Wnt signaling in vertebrates is to regulate planar cell polarity (PCP), the polarity axis that organizes the plane of a tissue. Cells at the invasive front of cellular aggregates exhibit elevated expression of genes associated with motility pathways, including Wnt/PCP signaling. Using microfluidic devices, we have previously shown the soluble non-canonical Wnt ligand, <i>WNT5A</i>, influencing the directional motion of ovarian cancer cells via the ROR2 receptor. We sought to determine whether asymmetrical localization of ROR2 could guide <i>WNT5A-</i>directed motility. <b>Methods:</b> A DNA sequence encoding a fusion protein of ROR2 with a C-terminal Emerald (Em) fluorescent protein tag was introduced into ES2 ovarian cancer cells. Aggregated spheroids of ES2 ROR2-Em cells were plated onto a fibronectin-coated surface, and time-lapse fluorescent images were obtained of the radial migration in the presence or absence of <i>WNT5A</i>. Based on the direction of motion, cells were divided into leading or trailing half for analysis of the localization of ROR2 protein. Data were presented as mean +/- SEM. For co-immunoprecipitation experiments, ES2 cells expressing a Myctagged ROR2 DNA construct were utilized, with Myc epitope used for pulldown. <b>Results:</b> In the absence of <i>WNT5A</i>, ROR2 was uniformly distributed among the leading and lagging edge of the migrating cells (46.9% +/-2.452 ROR2 localized on the leading edge). In the presence of <i>WNT5A</i>, ROR2 was preferentially located to the leading edge of migrating cells (64.6% +/- 3.908 ROR2 localized on the leading edge). <i>VANGL2</i>, a core component in planar cell polarity, formed Wnt-induced receptor complexes with ROR2 in developing limb buds and other tumor types. <i>VANGL2</i> had been shown to reside at the protrusive edge of leader cells. To address whether <i>VANGL2</i>-ROR2 complexes contributed to the leading-edge localization of ROR2 in ovarian cancer cells, co-IP experiments were performed. Complex formation of ROR2-<i>VANGL2</i> was present in ovarian cancer cells at baseline (<i>VANGL2</i>:ROR2 IP ratio 1), yet was increased in the presence of <i>WNT5A</i> (<i>VANGL2</i>:ROR2 IP ratio 1.5). <b>Conclusions:</b> We have previously shown that <i>WNT5A</i> promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as the degree of displacement in a ROR2 dependent manner. Asymmetric localization of Wnt/PCP core component complexes within leader and follower cells is crucial for cell motility, an intrinsic metastatic property. Our studies suggest that under the influence of <i>WNT5A</i>, ROR2 localizes to the leading edge of migrating cells. Additionally, ROR2 complexes with <i>VANGL2</i> in ovarian cancer cells, and this complex is enhanced in the presence of <i>WNT5A</i>. As <i>WNT5A</i> is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the microenvironment of ovarian cancer metastasis, disruption of the ROR2-<i>VANGL2</i> complex represents an attractive target for disrupting metastatic colonization of ovarian tumors.

Identification of a direct role of CPSF6 and a key cis‐acting motif in promoting TRIM9 proximal polyA site usage

Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3’ termini, is a rapidly expanding area of research that in recent studies has been linked to the mechanistic target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism. Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling and APA remains poorly understood. We previously leveraged our cTag‐PAPERCLIP technique to generate a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin ligase with a role in neurodevelopment, as a gene of interest. In this study, we further characterized the regulation of the mTOR‐induced TRIM9 APA shift observed in mouse neurons in vivo. Further study of the regulation of TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms, with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Additional study into the 3’UTR sequence elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence elements essential for CPSF6‐mediated regulation of the proximal TRIM9 PAS, we developed a RT‐qPCR PAS competition assay to quantify sequence‐mediated usage of PASs. Utilizing this assay, we assessed usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we generated constructs containing mutated UGUA sequences in order to ascertain the importance of the UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the TRIM9 proximal PAS. Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a reduction in TRIM9 proximal PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis‐acting motif in promoting TRIM9 proximal PAS usage. Furthermore, our results also suggest a possible link between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and neurodevelopment through TRIM9.

Regulation of Plant Immunity by Nuclear Membrane-Associated Mechanisms.

Unlike animals, plants do not have specialized immune cells and lack an adaptive immune system. Instead, plant cells rely on their unique innate immune system to defend against pathogens and coordinate beneficial interactions with commensal and symbiotic microbes. One of the major convergent points for plant immune signaling is the nucleus, where transcriptome reprogramming is initiated to orchestrate defense responses. Mechanisms that regulate selective transport of nuclear signaling cargo and chromatin activity at the nuclear boundary play a pivotal role in immune activation. This review summarizes the current knowledge of how nuclear membrane-associated core protein and protein complexes, including the nuclear pore complex, nuclear transport receptors, and the nucleoskeleton participate in plant innate immune activation and pathogen resistance. We also discuss the role of their functional counterparts in regulating innate immunity in animals and highlight potential common mechanisms that contribute to nuclear membrane-centered immune regulation in higher eukaryotes.

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery.

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Photoacoustic molecular imaging-escorted adipose photodynamic-browning synergy for fighting obesity with virus-like complexes.

Photodynamic therapy and adipose browning induction are two promising approaches to reverse obesity. The former strategy acts rapidly and locally, whereas the latter has a more gradual and widespread effect. Despite their complementarity, they have rarely been combined and imaged non-invasively in vivo. Here we introduce an adipose-targeting hepatitis B core protein complex that contains a traceable photosensitizer (ZnPcS4 (zinc phthalocyanine tetrasulfonate)) and a browning agent (rosiglitazone) that allows simultaneous photodynamic and browning treatments, with photoacoustic molecular imaging. After intravenous injection in obese mice, the complex binds specifically to white adipose tissues, especially those rich in blood supply, and drives adipose reduction thanks to the synergy of ZnPcS4 photodynamics and rosiglitazone browning. Using photoacoustic molecular imaging, we could monitor the changes induced by the treatment, which included complex activity, lipid catabolism and angiogenesis. Our findings demonstrate the anti-obesity potential of our feedback-based synergic regimen orchestrated by the targeted hepatitis B core complex.

Functional identification of Bombyx mori Atg13 in autophagy.

The autophagy process involves a series of autophagy-related (Atg) proteins, which are conserved in eukaryotes. ULK1/Atg1-ATG13/Atg13 is the core protein complex for autophagy initiation in response to nutrient and hormone signaling. However, how Atg13 is regulated to participate in autophagy is unclear in insects. Here in Bombyx mori, the variation of BmAtg13 was correlated with autophagy induced by steroid hormone 20-hydroxyecdysone (20E) or starvation. Developmental profiles from feeding to prepupal stage revealed that there were two bands of BmAtg13 protein detected by western blot analysis, therein the upper band was intensively decreased, while the lower band was significantly increased which was in accordance with its mRNA variation; and immunofluorescent staining indicated that BmAtg13 was nucleocytoplasmic translocated during larval-pupal metamorphosis when autophagy was dramatically induced. BmAtg13 knockdown and overexpression both inhibits autophagy. Besides, 20E treatment-induced BmAtg13 gene expression, while blocking 20E signaling transduction by knockdown of BmUsp reduced both gene expression and protein level of BmAtg13. These results reveal that BmAtg13 is required for 20E- and starvation-induced autophagy in B. mori, which provides the foundation for further related studies.

Thermal stability analyses of human PERIOD-2 C-terminal domain using dynamic light scattering and circular dichroism.

At the molecular level, the circadian clock is regulated by a time delayed transcriptional-translational feedback loop in which the core proteins interact with each other rhythmically to drive daily biological rhythms. The C-terminal domain of a key clock protein PER2 (PER2c) plays a critically important role in the loop, not only for its interaction with the binding partner CRY proteins but also for the CRY/PER complex’s translocation from the cytosol to the nucleus. Previous circular dichroism (CD) spectroscopic studies have shown that mouse PER2c (mPER2c) is less structured in solution by itself but folded into stable secondary structures upon interaction with mouse CRYs. To understand the stability and folding of human PER2c (hPER2c), we expressed and purified hPER2c. Three oligomerization forms of recombinant hPER2c were identified and thoroughly characterized through a combination of biochemical and biophysical techniques. Different to mPER2c, both thermal unfolding DLS and CD analyses suggested that all forms of hPER2c have very stable secondary structures in solution by themselves with melting temperatures higher than the physiological body temperature, indicating that hPER2c does not require CRY to fold. Furthermore, we examined the effects of EDTA, salt concentration, and a reducing agent on hPER2c folding and oligomerization. The ability of hPER2c forming oligomers reflects the potential role of hPER2c in the assembly of circadian rhythm core protein complexes.

Evaluating differences in growth, photosynthetic efficiency, and transcriptome of Asterarcys sp. SCS-1881 under autotrophic, mixotrophic, and heterotrophic culturing conditions

Autotrophic microalgae can assimilate organic carbon sources for the growth of heterotrophy and mixotrophy. In particular, the physiological and biochemical response of different microalgae species on autotrophy, heterotrophy, and mixotrophy are quite different. The aim of the present study was to determine differences in growth, biochemical composition, photosynthetic characteristics, and metabolic pathway under autotrophic, heterotrophic and mixotrophic culture conditions in an oleaginous microalga, Asterarcys sp. SCS-1881. The results showed that Asterarcys sp. can use glucose for mixotrophic growth, but cannot under strict heterotrophic growth. Mixotrophic cultures can significantly increase the biomass concentration and cell count of Asterarcys sp. The microalga accumulated more protein under mixotrophic conditions, but more carbohydrates and total lipids under autotrophic conditions. The microalga under mixotrophic culture conditions showed significantly reduced pigment content, PSII activity, and photoprotective ability, but increased the activity of PSI when compared to those under autotrophic conditions. Under mixotrophic conditions, Asterarcys sp. also showed enhanced glycolysis, photosynthetic carbon fixation, and pentose phosphate pathway and thus more ATP, carbon skeleton structure, and NADPH. Additionally, the biosynthesis of triacylglycerol, carbohydrates, and pigments was reduced under mixotrophic conditions and the core protein complexes of PSI and PSII were attenuated. Similarly, photosynthetic electron transport and many other pathways related to protein synthesis were enhanced under mixotrophic conditions when compared to the microalga cultured under autotrophic conditions. These biochemical changes may work to co-operatively ensure a more efficient supply of energy and carbon skeleton for rapid growth of the microalga under mixotrophic conditions when compared to those raised under autotrophic conditions.

Ciclopirox inhibits Hepatitis B Virus secretion by blocking capsid assembly

Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.2.15 cells. We find that ciclopirox, a synthetic antifungal agent, strongly inhibits HBV replication in cells and in mice by blocking HBV capsid assembly. The crystal structure of the HBV core protein and ciclopirox complex reveals a unique binding mode at dimer-dimer interfaces. Ciclopirox synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model. Therefore, orally-administered ciclopirox may provide a novel opportunity to combat chronic HBV infection by blocking HBV capsid assembly.

Respiration: Life Without Complex I

Eukaryotic life has developed a fascinating and highly optimized system for energy transduction: the mitochondrial respiratory chain. Typically composed of five core protein complexes, we now learn from two studies that plant hemi-parasites of the type Viscum cope without Complex I, the entry point of the classical respiratory system.

Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development.

In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem), cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

Comparative analysis of single‐cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells

Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells.

A common neighbor based technique to detect protein complexes in PPI networks.

Detection of protein complexes by analyzing and understanding PPI networks is an important task and critical to all aspects of cell biology. We present a technique called PROtein COmplex DEtection based on common neighborhood (PROCODE) that considers the inherent organization of protein complexes as well as the regions with heavy interactions in PPI networks to detect protein complexes. Initially, the core of the protein complexes is detected based on the neighborhood of PPI network. Then a merging strategy based on density is used to attach proteins and protein complexes to the core-protein complexes to form biologically meaningful structures. The predicted protein complexes of PROCODE was evaluated and analyzed using four PPI network datasets out of which three were from budding yeast and one from human. Our proposed technique is compared with some of the existing techniques using standard benchmark complexes and PROCODE was found to match very well with actual protein complexes in the benchmark data. The detected complexes were at par with existing biological evidence and knowledge.

Robust Asymmetric Localization of Planar Polarity Proteins Is Associated with Organization into Signalosome-like Domains of Variable Stoichiometry

SummaryIn developing epithelia, the core planar polarity proteins physically interact with each other and localize asymmetrically at opposite cell ends, forming intercellular complexes that link the polarity of neighboring cells. Using quantitative imaging to examine the composition of the core protein complex in vivo, we find that complex composition is unexpectedly plastic. The transmembrane proteins Frizzled and Flamingo form a stoichiometric nucleus in the complex, while the relative levels of the other four core proteins can vary independently. Exploring the functional consequences of this, we show that robust cell polarization is achieved over a range of complex stoichiometries but is dependent on maintaining appropriate levels of the components Frizzled and Strabismus. We propose that the core proteins assemble into signalosome-like structures, where stable association is not dependent on one-to-one interactions with binding partners, and signaling functions can act over a wide range of complex compositions.

Bark beetle controls epithelial morphogenesis by septate junction maturation in Drosophila

Epithelial tissues separate body compartments with different compositions. Tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates control the paracellular flow of molecules between these compartments. This epithelial barrier function of TJs and SJs must be stably maintained in tissue morphogenesis during cell proliferation and cell movement. Here, we show that Bark beetle (Bark), a putative transmembrane scavenger receptor-like protein, is essential for the maturation but not the establishment of SJs in Drosophila. Embryos that lack bark establish functional SJs, but due to rudimentary septae formation during subsequent embryonic development, these become non-functional. Furthermore, cell adhesion is impaired at the lateral cell membrane and the core protein complexes of SJs are mis-localised, but appear to form otherwise normally in such embryos. We propose a model in which Bark acts as a scaffold protein that mediates cell adhesion and mounting of SJ core complexes during cell rearrangement in tissue morphogenesis.

Regulation of Reversible Dissociation of LHCII from PSII by Phosphorylation in Plants

LHCII is a crucial light-harvesting pigment/protein complex in photosystem II (PSII) supercomplex. It also participates in the light energy redistribution between photosystems and in the photoprotection via its reversible dissociation with PSII and PSI (photosystem I). This reversible detachment of LHCII is regulated by phosphorylation of its own and PSII core protein. Under low light conditions, LHCII is phosphorylated and dissociated with PSII core protein complex and combined with PSI, which balances the excitation energy between PSII and PSI; Under high light environment, the phosphorylation of PSII core proteins makes LHCII detach from PSII. The dissociated LHCII presents in a free state, which involves in the thermal dissipation of excess excitation energy. During photodamage, dual phosphorylations of both PSII core proteins and LHCII complexes occur. The phosphorylation of D1 is conductive to the disintegration of photodamaged PSII and the cycle of repair. In this circumstance, the phosphorylation of LHCII is induced by reactive oxygen species (ROS) and then the phosphorylated LHCII migrates to PSI, into the repair cycle of damaged PSII. The ferredoxin (Fdr) and thioredoxin (Tdr) system may play a possible central role in the phosphorylation regulation on LHCII dissociation.

Re-Docking Scheme for Generating Near-Native Protein Complexes by Assembling Residue Interaction Fingerprints

Interaction profile method is a useful method for processing rigid-body docking. After the docking process, the resulting set of docking poses could be classified by calculating similarities among them using these interaction profiles to search for near-native poses. However, there are some cases where the near-native poses are not included in this set of docking poses even when the bound-state structures are used. Therefore, we have developed a method for generating near-native docking poses by introducing a re-docking process. We devised a method for calculating the profile of interaction fingerprints by assembling protein complexes after determining certain core-protein complexes. For our analysis, we used 44 bound-state protein complexes selected from the ZDOCK benchmark dataset ver. 2.0, including some protein pairs none of which generated near-native poses in the docking process. Consequently, after the re-docking process we obtained profiles of interaction fingerprints, some of which yielded near-native poses. The re-docking process involved searching for possible docking poses in a restricted area using the profile of interaction fingerprints. If the profile includes interactions identical to those in the native complex, we obtained near-native docking poses. Accordingly, near-native poses were obtained for all bound-state protein complexes examined here. Application of interaction fingerprints to the re-docking process yielded structures with more native interactions, even when a docking pose, obtained following the initial docking process, contained only a small number of native amino acid interactions. Thus, utilization of the profile of interaction fingerprints in the re-docking process yielded more near-native poses.

Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.