Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.

The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Designing photoaffinity tool compounds for the investigation of the DENV NS2B-NS3 protease allosteric binding pocket.

Dengue virus (DENV) infection still lacks specific antiviral therapy, making the NS2B-NS3 protease an attractive target for drug development. However, allosteric inhibitors that bind to a site other than the active site still need to be better understood. In this study, we designed and synthesised tool compounds for photoaffinity labelling (PAL) to investigate the binding site of allosteric inhibitors on the DENV protease. These tool compounds contained an affinity moiety, a photoreactive group, and a reporter tag for detection. Upon irradiation, the photoreactive group formed a covalent bond with the protease, allowing for binding site identification. SDS-PAGE-based assays confirmed the qualitative binding of the designed inhibitors to the allosteric pocket, and pull-down experiments validated the interaction. Tryptic protein digestion following liquid chromatography/mass spectrometry analysis further supported the binding of the inhibitor to the proposed pocket revealing photo-attachment to an NS3 loop close to the C-terminus. These results enhance our understanding of allosteric inhibitors and their mechanism of action against the DENV protease. The developed tool compounds and PAL are potent tools for future drug discovery efforts and investigations targeting the DENV protease.

Proteome changes of fibroblasts and endothelial cells upon incubation with human cytomegalovirus subviral Dense Bodies.

Human cytomegalovirus (HCMV) is a pathogen of high medical relevance. Subviral Dense Bodies (DB) were developed as a vaccine candidate to ameliorate the severe consequences of HCMV infection. Development of such a candidate vaccine for human application requires detailed knowledge of its interaction with the host. A comprehensive mass spectrometry (MS)- based analysis was performed regarding the changes in the proteome of cell culture cells, exposed to DB.

Cell-of-origin-specific proteomics of extracellular vesicles.

The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native purification of intact EVs from a target cell, via a bioorthogonal tetrazine ligation, leading to copurification of the largely unlabeled EV proteome from the same cell. SIEVE enables capture of EV proteins at levels comparable with those obtained by antibody-based methods, which capture all EVs regardless of cellular origin, and at levels 20× higher than direct capture of SORT-labeled proteins. Using proteomic analysis, we analyze nonlabeled cargo proteins of EVs and show that the enhanced sensitivity of SIEVE allows for unbiased and comprehensive analysis of EV proteins from subpopulations of cells as well as for cell-specific EV proteomics in complex coculture systems. SIEVE can be applied with high efficiency in a diverse range of existing model systems for cell-cell communication and has direct applications for cell-of-origin EV analysis and for protein biomarker discovery.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex.

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration.

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

Cardiovascular diseases are one of the world's biggest killers. Even for patients who survive a heart attack, recovery can be difficult. This is because ­ unlike some amphibians and fish ­ humans lack the ability to produce enough new heart muscle cells to replace damaged tissue after a heart injury. In other words, the human heart cannot repair itself. Molecules known as messenger RNA (mRNA) carry the 'instructions' from the DNA inside the cell nucleus to its protein-making machinery in the cytoplasm of the cell. These messenger molecules can also be altered by different enzymes that attach or remove chemical groups. These modifications can change the stability of the mRNA, or even 'silence' it altogether by stopping it from interacting with the protein-making machinery, thus halting production of the protein it encodes. For example, a protein called Mettl3 can attach a methyl group to a specific part of the mRNA, causing a reversible mRNA modification known as m⁶A. This type of alteration has been shown to play a role in many conditions, including heart disease, but it has been unclear whether m⁶A could also be important for the regeneration of heart tissue. To find out more, Jiang, Liu, Chen et al. studied heart injury in mice of various ages. Newborn mice can regenerate their heart muscle for a short time, but adult mice lack this ability, which makes them a useful model to study heart disease. Analyses of the proteins and mRNAs in mouse heart cells confirmed that both Mettl3 and m⁶A-modified mRNAs were present. The amount of each also increased with age. Next, experiments in genetically manipulated mice revealed that removing Mettl3 greatly improved tissue repair after heart injury in both newborn and adult mice. In contrast, mouse hearts that produced abnormally high quantities of Mettl3 were unable to regenerate ­ even if the mice were young. Moreover, a detailed analysis of gene activity revealed that Mettl3 was suppressing heart regeneration by decreasing the production of a growth-promoting protein called FGF16. These results reveal a key biological mechanism controlling the heart's ability to repair itself after injury. In the future, Jiang et al. hope that Mettl3 can be harnessed for new, effective therapies to promote heart regeneration in patients suffering from heart disease.

Significant enhancement of scintillation performance by inducing oxygen vacancies in alkali metal ion (A+ = Li+, Na+, K+)-incorporated (Lu, Sc)BO3:Ce.

The incorporation of Sc3+ can stabilize calcite-phase LuBO3:Ce3+ to grow large-sized single crystals but leads to the significant degradation of scintillation performance. In the present work, alkali metal ion (A+ = Li+, Na+, K+)-incorporated (Lu, A, Sc)BO3:Ce was rapidly synthesized in batches via a high-throughput sol-gel method. The aliovalent substitution of Lu3+ with A+ is balanced by the generation of oxygen vacancies by forming complexes. Thanks to the increased oxygen vacancies, the luminescence and XEL intensity of (Lu, Li, Sc)BO3:Ce are significantly enhanced by 2.2 times and 1.9 times, respectively. Further, the incorporation of A+ is attributed to the improved transition efficiency of charge carriers. The prepared scintillation screen fabricated with LASBO:Ce and PMMA shows that the spatial resolution can reach 8.6 lp mm-1, indicating its potential application in efficient and low-cost non-destructive X-ray detection. This work is of great significance in improving the luminescence and scintillation performance of (Lu, Sc)BO3:Ce single crystals and thin films and their application in the scintillation field.

Highly Reactive Cyclic Carbonates with a Fused Ring toward Functionalizable and Recyclable Polycarbonates.

Monomer design plays an important role in the development of polymers with desired thermal properties and chemical recyclability. Here we prepared a class of seven-membered ring carbonates containing trans-cyclohexyl fused rings. These monomers showed excellent activity for ring-opening polymerization (ROP) with turnover frequency (TOF) up to 6 × 105 h-1 and catalyst loading down to 50 ppm, which yielded high-molecular-weight polycarbonates (Mn up to 673 kg/mol) with great thermostability (Td > 300 °C). Ultimately, the resulting polycarbonates can completely depolymerize into their corresponding cyclic dimers that can repolymerize to synthesize the starting polymers in moderate yields, demonstrating a potential route to achieve chemical recycling. Postfunctionalization of the unsaturated polycarbonate was conducted through cross-linking reaction and "click" reaction under UV irradiation.

Development of a [2 + 2]-Nitroso/Alkene Cycloaddition Using Sodium Tetrakis[3,5-bis(trifluoromethyl)phenyl]borate Catalyst: Controlled Chemoselectivity of Two Equilibrating Isomeric Intermediates.

Sodium tetrakis[3,5-bis(trifluoromethyl)-phenyl]borate (NaBArF) catalyzes the [2 + 2] cycloaddition of 1,4-disubstituted cyclopenta-1,3-dien-2-yl esters with nitrsobenzene in toluene, affording two isolable regioisomers of 6-oxa-7-azabicyclo[3.2.0] heptanes, which thermally rearrange into the same 4-aminocyclopent-1-en-3-ones. In the case of 4-substituted cyclopenta-1,3-dien-2-yl esters, their initial [2 + 2] cycloaddition intermediates undergo a rapid ring expansion to afford six-membered piperidone derivatives efficiently.

Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules.

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

Correction: Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells.

[This corrects the article DOI: 10.1371/journal.pgen.1004103.].

Enhancing the mechanical and thermal properties of polypropylene composite by encapsulating styrene acrylonitrile with ammonium polyphosphate.

BACKGROUNDS: In recent decades, incorporating polypropylene (PP) within flame retardants has proved to be an effective method of improving the thermal stabilities of PP, but too much adversely affects the mechanical properties of this polymer materials. Herein we report a novel multifunctional flame retardant, (styrene acrylonitrile)-(titanate-modified ammonium polyphosphate) (SAN-TAPP), to simultaneously improve the mechanical properties and thermal stability of PP composites. METHODS: SAN-TAPP was synthesized by encapsulating SAN resins with functional titanate-modified APP (TAPP) and subsequently was incorporated into PP by a melt-blending process. The phase characteristics and morphology of SAN-TAPP were investigated, and the mechanical properties and thermal stability of different content of PP/SAN-TAPP composites were studied. RESULTS: The results showed that the TAPP was almost entirely wrapped in the SAN resins and PP/SAN-TAPP composites exhibited the sea-island morphology. For the mechanical properties, the impact strength of PP/SAN-TAPP composite was significantly improved, especially 15 wt% SAN-TAPP filled PP/SAN-TAPP composite exhibiting 2.17 times higher than that of pure PP. And the tensile strength and modulus also increased by addition of SAN-TAPP. For the thermal stabilities, melting temperatures (Tm) and residual char yield were improved. Furthermore, the LOI value of PP/SAN-TAPP composites increased from 19.8 to 27.5%; The 15 and 20 wt% SAN-TAPP filled in PP/SAN-TAPP composites passed the V-2 test of UL-94, and exerted the similar effect on the flame retardancy to TAPP with the same loading. CONCLUSIONS: These results revealed that a novel PP/SAN-TAPP composites with synthetically enhancement on the mechanical properties, thermal stabilities and flame retardancy, suggesting a strong correlation between the phase structure, mechanical and thermal properties.

Centriolar satellites are acentriolar assemblies of centrosomal proteins.

Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)-based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole-cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.

Evolutionary plasticity in the innate immune function of Akirin.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

Kinase-controlled phase transition of membraneless organelles in mitosis.

Liquid-liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis.

In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics.

Studying protein interactions in whole organisms is fundamental to understanding development. Here, we combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development. Our network reflects known biology and is highly enriched in functionally relevant interactions. To demonstrate the utility of the map, we looked for new regulators of P granule dynamics and found that GEI-12, a novel binding partner of the DYRK family kinase MBK-2, is a key regulator of P granule formation and germline maintenance. Our data corroborate a recently proposed model in which the phosphorylation state of GEI-12 controls P granule dynamics. In addition, we find that GEI-12 also induces granule formation in mammalian cells, suggesting a common regulatory mechanism in worms and humans. Our results show that in vivo interaction proteomics provides unique insights into animal development.

In vivo stable isotope labeling by amino acids in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the most widely used and well-studied model organisms in biology and therefore a promising tool for quantitative proteomics. Here, we describe a method to label D. melanogaster with stable isotope labeled amino acids in vivo. Feeding flies with heavy lysine labeled yeast cells leads to virtually complete heavy labeling already in the first filial generation. The approach is simple, fast, and cost-effective, which makes SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.