TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.

Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity.

Interleukin-2 (IL-2) and downstream transcription factor STAT5 are important for maintaining regulatory T (Treg) cell homeostasis and function. Treg cells can respond to low IL-2 levels, but the mechanisms of STAT5 activation during partial IL-2 deficiency remain uncertain. We identified the serine-threonine kinase Mst1 as a signal-dependent amplifier of IL-2-STAT5 activity in Treg cells. High Mst1 and Mst2 (Mst1-Mst2) activity in Treg cells was crucial to prevent tumor resistance and autoimmunity. Mechanistically, Mst1-Mst2 sensed IL-2 signals to promote the STAT5 activation necessary for Treg cell homeostasis and lineage stability and to maintain the highly suppressive phosphorylated-STAT5+ Treg cell subpopulation. Unbiased quantitative proteomics revealed association of Mst1 with the cytoskeletal DOCK8-LRCHs module. Mst1 deficiency limited Treg cell migration and access to IL-2 and activity of the small GTPase Rac, which mediated downstream STAT5 activation. Collectively, IL-2-STAT5 signaling depends upon Mst1-Mst2 functions to maintain a stable Treg cell pool and immune tolerance.

Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours 1 . Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified 2 , the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α- dendritic cells. Dendritic cell-specific deletion of Mst1/2-but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling-disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α- dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.

Co2Mo6S8 Catalyzes Nearly Exclusive Electrochemical Nitrate Conversion to Ammonia with Enzyme-like Activity.

Electrocatalytic nitrate to ammonia conversion is a key reaction for energy and environmental sustainability. This reaction involves complex multi electron and proton transfer steps, and is impeded by the lack of catalyst for promoting both reactivity and ammonia selectivity. Here, we demonstrate active motifs based on the Chevrel phase Co2Mo6S8 exhibit an enzyme-like high turnover frequency of ∼95.1 s-1 for nitrate electroreduction to ammonia. We reveal strong synergy of multiple binding sites on this catalyst, such that the ligand effect of Co steers Had* toward hydrogenation other than hydrogen evolution, the ensemble effect of Co, and the spatial confinement effect that promote the full hydrogenation of NOx to ammonia without N-N coupling. The catalyst exhibits almost exclusive ammonia conversion with a Faradaic efficiency of 97.1% and ammonia yielding rate of 115.5 mmol·gcat-1·h-1 in neutral electrolytes. The high activity was also confirmed in electrolytes with dilute nitrate and high chloride concentrations.

Effect of Poly(propylene carbonate) on Properties of Polylactic Acid-Based Composite Films.

To enrich the properties of polylactic acid (PLA)-based composite films and improve the base degradability, in this study, a certain amount of poly(propylene carbonate) (PPC) was added to PLA-based composite films, and PLA/PPC-based composite films were prepared by melt blending and hot-press molding. The effects of the introduction of PPC on the composite films were analyzed through in-depth studies on mechanical properties, water vapor and oxygen transmission rates, thermal analysis, compost degradability, and bacterial inhibition properties of the composite films. When the introduction ratio coefficient of PPC was 30%, the tensile strength of the composite film increased by 19.68%, the water vapor transmission coefficient decreased by 14.43%, and the oxygen transmission coefficient decreased by 18.31% compared to that of the composite film without PPC, the cold crystallization temperature of the composite film increased gradually from 96.9 °C to 104.8 °C, and PPC improved the crystallization ability of composite film. The degradation rate of the composite film with PPC increased significantly compared to the previous one, and the degradation rate increased with the increase in the PPC content. The degradation rate was 49.85% and 46.22% faster on average than that of the composite film without PPC when the degradation was carried out over 40 and 80 days; the composite film had certain inhibition, and the maximum diameter of the inhibition circle was 2.42 cm. This study provides a strategy for the development of PLA-based biodegradable laminates, which can promote the application of PLA-based laminates in food packaging.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

Photocatalytic CO2 Reduction with Dissolved Carbonates and Near-Zero CO2(aq) by Employing Long-Range Proton Transport.

Photocatalytic CO2 reduction (CO2R) in ∼0 mM CO2(aq) concentration is challenging but is relevant for capturing CO2 and achieving a circular carbon economy. Despite recent advances, the interplay between the CO2 catalytic reduction and the oxidative redox processes that are arranged on photocatalyst surfaces with nanometer-scale distances is less studied. Specifically, mechanistic investigation on interdependent processes, including CO2 adsorption, charge separation, long-range chemical transport (∼100 nm distance), and bicarbonate buffer speciation, involved in photocatalysis is urgently needed. Photocatalytic CO2R in ∼0 mM CO2(aq), which has important applications in integrated carbon capture and utilization (CCU), has rarely been studied. Using 0.1 M KHCO3 (aq) of pH 7 but without continuously bubbling CO2, we achieved ∼0.1% solar-to-fuel conversion efficiency for CO production using Ag@CrOx nanoparticles that are supported on a coating-protected GaInP2 photocatalytic panel. CO is produced at ∼100% selectivity with no detectable H2, even with copious protons co-generated nearby. CO2 flux to the Ag@CrOx CO2R sites enhances CO2 adsorption, probed by in situ Raman spectroscopy. CO is produced with local protonation of dissolved inorganic carbon species in a pH as high as 11.5 when using fast electron donors such as ethanol. Isotopic labeling using KH13CO3 was used to confirm the origin of CO from the bicarbonate solution. We then employed COMSOL Multiphysics modeling to simulate the spatial and temporal pH variation and the local concentrations of bicarbonates and CO2(aq). We found that light-driven CO2R and CO2 reactive transport are mutually dependent, which is important for further understanding and manipulating CO2R activity and selectivity. This study enables direct bicarbonate utilization as the source of CO2, thereby achieving CO2 capture and conversion without purifying and feeding gaseous CO2.

Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFß signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFß-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Regulation of gene expression by miR-144/451 during mouse erythropoiesis.

The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.

Combination of medical and health care based on digital smartphone-powered photochemical dongle for renal function management.

Currently, the global healthcare market is increasing at high speed with the impendent global aging issue. Healthcare Industry 4.0 has emerged as an efficient solution towards the aging issue since it was integrated with ubiquitous medical sensors, big health processing platform, high bandwidth, speed technologies, and medical services, etc. It is believed to fulfil the requirement of the tremendously growing health market. The acquisition of medical data acts as the dominant precondition to implement the Healthcare Industry 4.0. In the same way, the widely available smartphone could serve as the best biomedical information collect station. In this study, a smartphone-powered photochemical dongle is demonstrated to precisely estimate blood creatinine from the fingertip blood, which works as a highly compact reflectance spectral analyzer with an enzymatically dry chemical test strip. Comparing with conventional laboratory facility for the evaluation and treatment of chronic kidney disease (CKD), it implemented the platform of point care with agreed accuracy. In order to estimate the efficiency of treatment and recovery of the CKD, the proposed photochemical dongle would provide a flexible and rapid platform for point of care. Furthermore, the proposed measured technology is very promising method for remote CKD management.

27-Plex Tandem Mass Tag Mass Spectrometry for Profiling Brain Proteome in Alzheimer's Disease.

Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem mass spectrometry (MS/MS) for peptide quantification and then apply the method to profile the complex human brain proteome of Alzheimer's disease (AD). The 27-plex method combines multiplexed capacities of the 11-plex and the 16-plex TMT, as the peptides labeled by the two TMT sets display different mass and hydrophobicity, which can be well separated in LC-MS/MS. We first systematically optimized the protocol for the newly developed 16-plex TMT, including labeling reaction, desalting, and MS conditions, and then directly compared the 11-plex and 16-plex methods by analyzing the same human AD samples. Both methods yielded similar proteome coverage, analyzing >100 000 peptides in >10 000 human proteins. Furthermore, the 11-plex and 16-plex samples were mixed for a 27-plex assay, resulting in more than 8000 protein measurements within the same MS time. The 27-plex results are highly consistent with those of the individual 11-plex and 16-plex TMT analyses. We also used these proteomics data sets to compare the AD brain with the nondementia controls, discovering major AD-related proteins and revealing numerous novel protein alterations enriched in the pathways of amyloidosis, immunity, mitochondrial, and synaptic functions. Overall, our data strongly demonstrate that this new 27-plex strategy is highly feasible for routine large-scale proteomic analysis.

Deep undepleted human serum proteome profiling toward biomarker discovery for Alzheimer's disease.

BACKGROUND: Blood-based protein measurement is a routine practice for detecting biomarkers in human disease. Comprehensive profiling of blood/plasma/serum proteome is a challenge due to an extremely large dynamic range, as exemplified by a small subset of highly abundant proteins. Antibody-based depletion of these abundant proteins alleviates the problem but introduces experimental variations. We aimed to establish a method for direct profiling of undepleted human serum and apply the method toward biomarker discovery for Alzheimer's disease (AD), as AD is the most common form of dementia without available blood-based biomarkers in clinic. METHODS: We present an ultra-deep analysis of undepleted human serum proteome by combining the latest 11-plex tandem-mass-tag (TMT) labeling, exhaustive two-dimensional liquid chromatography (LC/LC) fractionation (the 1st LC: 3 h for 180 fractions, and the 2nd LC: 3 h gradient per fraction), coupled with high resolution tandem mass spectrometry (MS/MS). AD (n = 6) and control (n = 5) sera were analyzed in this pilot study. In addition, we implemented a multiplexed targeted LC-MS3 method (TOMAHAQ) for the validation of selected target proteins. RESULTS: The TMT-LC/LC-MS/MS platform is capable of analyzing 4826 protein components (4368 genes), covering at least 6 orders of magnitude in dynamic range, representing one of the deepest serum proteome analysis. We defined intra- and inter- group variability in the AD and control groups. Statistical analysis revealed differentially expressed proteins in AD (26 decreased and 4 increased). Notably, these altered proteins are enriched in the known pathways of mitochondria, fatty acid beta oxidation, and AGE/RAGE. Finally, we set up a TOMAHAQ method to confirm the decrease of PCK2 and AK2 in our AD samples. CONCLUSIONS: Our results show an ultra-deep serum discovery study by TMT-LC/LC-MS/MS, and a validation experiment by TOMAHAQ targeted LC-MS3. The MS-based discovery and validation methods are of general use for biomarker discovery from complex biofluids (e.g. serum proteome). This pilot study also identified deregulated proteins, in particular proteins associated with mitochondrial function in the AD serum samples. These proteins may serve as novel AD candidate biomarkers.

Mutant LRRK2 mediates peripheral and central immune responses leading to neurodegeneration in vivo.

Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene result in late-onset Parkinson's disease. The incomplete penetrance of LRRK2 mutations in humans and LRRK2 murine models of Parkinson's disease suggests that the disease may result from a complex interplay of genetic predispositions and persistent exogenous insults. Since neuroinflammation is commonly associated with the pathogenesis of Parkinson's disease, we examine a potential role of mutant LRRK2 in regulation of the immune response and inflammatory signalling in vivo. Here, we show that mice overexpressing human pathogenic LRRK2 mutations, but not wild-type mice or mice overexpressing human wild-type LRRK2 exhibit long-term lipopolysaccharide-induced nigral neuronal loss. This neurodegeneration is accompanied by an exacerbated neuroinflammation in the brain. The increased immune response in the brain of mutant mice subsequently has an effect on neurons by inducing intraneuronal LRRK2 upregulation. However, the enhanced neuroinflammation is unlikely to be triggered by dysfunctional microglia or infiltrated T cells and/or monocytes, but by peripheral circulating inflammatory molecules. Analysis of cytokine kinetics and inflammatory pathways in the peripheral immune cells demonstrates that LRRK2 mutation alters type II interferon immune response, suggesting that this increased neuroinflammatory response may arise outside the central nervous system. Overall, this study suggests that peripheral immune signalling plays an unexpected-but important-role in the regulation of neurodegeneration in LRRK2-associated Parkinson's disease, and provides new targets for interfering with the onset and progression of the disease.

Target-Decoy-Based False Discovery Rate Estimation for Large-Scale Metabolite Identification.

Metabolite identification is a crucial step in mass spectrometry (MS)-based metabolomics. However, it is still challenging to assess the confidence of assigned metabolites. We report a novel method for estimating the false discovery rate (FDR) of metabolite assignment with a target-decoy strategy, in which the decoys are generated through violating the octet rule of chemistry by adding small odd numbers of hydrogen atoms. The target-decoy strategy was integrated into JUMPm, an automated metabolite identification pipeline for large-scale MS analysis and was also evaluated with two other metabolomics tools, mzMatch and MZmine 2. The reliability of FDR calculation was examined by false data sets, which were simulated by altering MS1 or MS2 spectra. Finally, we used the JUMPm pipeline coupled to the target-decoy strategy to process unlabeled and stable-isotope-labeled metabolomic data sets. The results demonstrate that the target-decoy strategy is a simple and effective method for evaluating the confidence of high-throughput metabolite identification.

Isotope Labeling-Assisted Evaluation of Hydrophilic and Hydrophobic Liquid Chromatograph-Mass Spectrometry for Metabolomics Profiling.

High throughput untargeted metabolomics usually relies on complementary liquid chromatography-mass spectrometry (LC-MS) methods to expand the coverage of diverse metabolites, but the integration of those methods is not fully characterized. We systematically investigated the performance of hydrophilic interaction liquid chromatography (HILIC)-MS and nanoflow reverse-phase liquid chromatography (nRPLC)-MS under 8 LC-MS settings, varying stationary phases (HILIC and C18), mobile phases (acidic and basic pH), and MS ionization modes (positive and negative). Whereas nRPLC-MS optimization was previously reported, we found in HILIC-MS (2.1 mm × 150 mm) that the optimal performance was achieved in a 90 min gradient with 100 µL/min flow rate by loading metabolite extracts from 2 mg of cell/tissue samples. Since peak features were highly compromised by contaminants, we used stable isotope labeled yeast to enhance formula identification for comparing different LC-MS conditions. The 8 LC-MS settings enabled the detection of a total of 1050 formulas, among which 78%, 73%, and 62% formulas were recovered by the best combination of 4, 3, and 2 LC-MS settings, respectively. Moreover, these yeast samples were harvested in the presence or absence of nitrogen starvation, enabling quantitative comparisons of altered formulas and metabolite structures, followed by validation with selected synthetic metabolites. The results revealed that nitrogen starvation downregulated amino acid components but upregulated uridine-related metabolism. In summary, this study introduces a thorough evaluation of hydrophilicity and hydrophobicity based LC-MS and provides information for selecting complementary settings to balance throughput and efficiency during metabolomics experiments.

Physicochemical evolutions of starch/poly (lactic acid) composite biodegraded in real soil.

Plastic pollution is a major environmental problem and the waste disposal is a challenge in this case. Poly (lactic acid) (PLA) based biodegradable materials is one of the most attractive polymers which can fulfill the current demand. In this work, the degradation of starch/PLA composite was investigated in real soil environment. The weight loss results demonstrated that the degradation rate of PLA could be accelerated by starch. Scanning electrical microscopy (SEM) and Fourier transform infrared (FTIR) results showed that the samples degraded faster with the presence of starch. The mechanical strengths had an abrupt decrease for the starch/PLA composite while that of PLA only decreased in a low degree. The distribution of carboxyl group intensity and carbon atomic percent reflected the heterogeneity of biodegradation for starch/PLA composite in soil. Moreover, the variation of internal carbon atomic percent was higher than that on the surface, demonstrating that the degradation of starch/PLA composite was bulk degradation. Based on the role of starch played in starch/PLA composite and the physicochemical performance evolutions during biodegradation, it should create a scientific basis for people interested in studying the biodegradation of PLA, and provide some knowledge about controlling the biodegradation rate of PLA through adjusting the content of starch in the composite.

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth.

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.

Enhanced Performance of Si MIS Photocathodes Containing Oxide-Coated Nanoparticle Electrocatalysts.

Electrodepositing low loadings of metallic nanoparticle catalysts onto the surface of semiconducting photoelectrodes is a highly attractive approach for decreasing catalyst costs and minimizing optical losses. However, securely anchoring nanoparticles to the photoelectrode surface can be challenging-especially if the surface is covered by a thin insulating overlayer. Herein, we report on Si-based photocathodes for the hydrogen evolution reaction that overcome this problem through the use of a 2-10 nm thick layer of silicon oxide (SiOx) that is deposited on top of Pt nanoparticle catalysts that were first electrodeposited on a 1.5 nm SiO2|p-Si(100) absorber layer. Such insulator-metal-insulator-semiconductor (IMIS) photoelectrodes exhibit superior durability and charge transfer properties compared to metal-insulator-semiconductor (MIS) control samples that lacked the secondary SiOx overlayer. Systematic investigation of the influence of particle loading, SiOx layer thickness, and illumination intensity suggests that the SiOx layer possesses moderate conductivity, thereby reducing charge transfer resistance associated with high local tunneling current densities between the p-Si and Pt nanoparticles. Importantly, the IMIS architecture is proven to be a highly effective approach for stabilizing electrocatalytic nanoparticles deposited on insulating overlayers without adversely affecting mass transport of reactant and product species associated with the hydrogen evolution reaction.