MAJIN Links Telomeric DNA to the Nuclear Membrane by Exchanging Telomere Cap.

In meiosis, telomeres attach to the inner nuclear membrane (INM) and drive the chromosome movement required for homolog pairing and recombination. Here, we address the question of how telomeres are structurally adapted for the meiotic task. We identify a multi-subunit meiotic telomere-complex, TERB1/2-MAJIN, which takes over telomeric DNA from the shelterin complex in mouse germ cells. TERB1/2-MAJIN initially assembles on the INM sequestered by its putative transmembrane subunit MAJIN. In early meiosis, telomere attachment is achieved by the formation of a chimeric complex of TERB1/2-MAJIN and shelterin. The chimeric complex matures during prophase into DNA-bound TERB1/2-MAJIN by releasing shelterin, forming a direct link between telomeric DNA and the INM. These hierarchical processes, termed "telomere cap exchange," are regulated by CDK-dependent phosphorylation and the DNA-binding activity of MAJIN. Further, we uncover a positive feedback between telomere attachment and chromosome movement, revealing a comprehensive regulatory network underlying meiosis-specific telomere function in mammals.

Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State.

MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.

Tropomyosin induces the synthesis of magnesian calcite in sea urchin spines.

Calcium carbonate is present in many biominerals, including in the exoskeletons of crustaceans and shells of mollusks. High Mg-containing calcium carbonate was synthesized by high temperatures, high pressures or high molecular organic matter. For example, biogenic high Mg-containing calcite is synthesized under strictly controlled Mg concentration at ambient temperature and pressure. The spines of sea urchins consist of calcite, which contain a high percentage of magnesium. In this study, we investigated the factors that increase the magnesium content in calcite from the spines of the sea urchin, Heliocidaris crassispina. X-ray diffraction and inductively coupled plasma mass spectrometry analyses showed that sea urchin spines contain about 4.8% Mg. The organic matrix extracted from the H. crassispina spines induced the crystallization of amorphous phase and synthesis of magnesium-containing calcite, while amorphous was synthesized without SUE (sea urchin extract). In addition, aragonite was synthesized by SUE treated with protease-K. HC tropomyosin was specifically incorporated into Mg precipitates. Recombinant HC-tropomyosin induced calcite contained 0.1-2.5% Mg synthesis. Western blotting of sea urchin spine extracts confirmed that HC tropomyosin was present in the purple sea urchin spines at a protein weight ratio of 1.5%. These results show that HC tropomyosin is one factor that increases the magnesium concentration in the calcite of H. crassispina spines.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant.

Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

SIPA1L1/SPAR1 Interacts with the Neurabin Family of Proteins and is Involved in GPCR Signaling.

Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.

Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

Dispersion Function of a Protein, DP-1, Identified in Collimonas sp. D-25, for the Synthesis of Gold Nanoparticles.

Collimonas sp. (D-25), found in the soil of Akita Prefecture, is a gram-negative bacterium with the ability to synthesize gold nanoparticles (AuNPs). During the synthesis of AuNPs, one specific protein (DP-1) was found to have disappeared in the sonicated solution of the bacterium. Recombinant DP-1 (rDP-1) from Escherichia coli BL21 (DE3) was used to study the effect of DP-1 on the synthesis of AuNPs. AuNPs synthesized with rDP-1 result in small, stabilized nanoparticles. AuNPs synthesized by DP-1 retained the stability of both the dispersion and nano-size particles under high salt concentrations. Isothermal titration calorimetry was employed to investigate the bonding ratio of rDP-1 to AuNPs. Several thousand rDP-1 proteins are attached to the surface of an AuNP to form a protein corona containing multiple layers. These results suggest that DP-1 obtained from D-25 has a size and stability control function during AuNP synthesis.

DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC.

The piRNA amplification pathway in Bombyx is operated by Ago3 and Siwi in their piRISC form. The DEAD-box protein, Vasa, facilitates Ago3-piRISC production by liberating cleaved RNAs from Siwi-piRISC in an ATP hydrolysis-dependent manner. However, the Vasa-like factor facilitating Siwi-piRISC production along this pathway remains unknown. Here, we identify DEAD-box polypeptide 43 (DDX43) as the Vasa-like protein functioning in Siwi-piRISC production. DDX43 belongs to the helicase superfamily II along with Vasa, and it contains a similar helicase core. DDX43 also contains a K-homology (KH) domain, a prevalent RNA-binding domain, within its N-terminal region. Biochemical analyses show that the helicase core is responsible for Ago3-piRISC interaction and ATP hydrolysis, while the KH domain enhances the ATPase activity of the helicase core. This enhancement is independent of the RNA-binding activity of the KH domain. For maximal DDX43 RNA-binding activity, both the KH domain and helicase core are required. This study not only provides new insight into the piRNA amplification mechanism but also reveals unique collaborations between the two domains supporting DDX43 function within the pathway.

Glycolaldehyde is an endogenous source of lysine N-pyrrolation.

Lysine N-pyrrolation converts lysine residues to Nϵ-pyrrole-l-lysine (pyrK) in a covalent modification reaction that significantly affects the chemical properties of proteins, causing them to mimic DNA. pyrK in proteins has been detected in vivo, indicating that pyrrolation occurs as an endogenous reaction. However, the source of pyrK remains unknown. In this study, on the basis of our observation in vitro that pyrK is present in oxidized low-density lipoprotein and in modified proteins with oxidized polyunsaturated fatty acids, we used LC-electrospray ionization-MS/MS coupled with a stable isotope dilution method to perform activity-guided separation of active molecules in oxidized lipids and identified glycolaldehyde (GA) as a pyrK source. The results from mechanistic experiments to study GA-mediated lysine N-pyrrolation suggested that the reactions might include GA oxidation, generating the dialdehyde glyoxal, followed by condensation reactions of lysine amino groups with GA and glyoxal. We also studied the functional significance of GA-mediated lysine N-pyrrolation in proteins and found that GA-modified proteins are recognized by apolipoprotein E, a binding target of pyrrolated proteins. Moreover, GA-modified proteins triggered an immune response to pyrrolated proteins, and monoclonal antibodies generated from mice immunized with GA-modified proteins specifically recognized pyrrolated proteins. These findings reveal that GA is an endogenous source of DNA-mimicking pyrrolated proteins and may provide mechanistic insights relevant for innate and autoimmune responses associated with glucose metabolism and oxidative stress.

The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis.

Long non-coding RNAs (lncRNAs) are aberrantly expressed in many disease conditions, including cancer. Accumulating evidence indicates that some lncRNAs may play critical roles in cancer progression and metastasis. Here, we identify a set of lncRNAs that are upregulated in metastatic subpopulations isolated from colon cancer HCT116 cells in vivo and show that one of these lncRNAs, which we name CALIC, is required for the metastatic activity of colon cancer cells. We show that CALIC associates with the RNA-binding protein hnRNP-L and imparts specificity to hnRNP-L-mediated gene expression. Furthermore, we demonstrate that the CALIC/hnRNP-L complex upregulates the tyrosine kinase receptor AXL and that knockdown of CALIC or AXL using shRNA in colon cancer cells attenuates their ability to form metastases in mice. These results suggest that the CALIC/hnRNP-L complex enhances the metastatic potential of colon cancer cells.

Characterization of the chalky layer-derived EGF-like domain-containing protein (CgELC) in the pacific oyster, Crassostrea gigas.

The shells of the Pacific oyster Crassostrea gigas contain calcite crystals with three types of microstructures: prismatic, chalky, and foliated layers. Many shell matrix proteins were annotated from the shells of C. gigas; however, it is unclear which SMPs play important roles in their shell mineralization. The matrix proteins have never been reported from the chalky layer. In this study, we identified a chalky layer-derived EGF-like domain-containing protein (CgELC) from the chalky layer of C. gigas shells. The gene sequence of the CgELC was encoded under CGI_ 10,017,544 of the C. gigas genome database. Only peptide fragments in the N-terminal region of CGI_ 10,017,544 were detected by LC-MS/MS analyses, suggesting that CGI_ 10,017,544 was digested at the predicted protease digestion dibasic site by post-translational modification to become a mature CgELC protein. We produced three types of CgELC recombinant proteins, namely, the full length CgELC, as well as the N-terminal and C-terminal parts of CgELC (CgELC-N or -C, respectively), for in vitro crystallization experiments. In the presence of these recombinant proteins, the aggregation of polycrystalline calcite was observed. Some fibrous organic components seemed to be incorporated into the calcite crystals in the presence of the r-CgELC protein. We also noted different features in the crystallization between CgELC-N and CgELC-C; some crystals were inhibited crystal plane formation and contained many columnar prisms inside the crystals (CgELC-N) and formed numerous holes on their surfaces (CgELC-C). These results suggest that CgELC is involved in crystal aggregation and incorporated into calcite crystals.

SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity.

Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma.

Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1.

Many long noncoding RNAs (lncRNAs) are reported to be dysregulated in human cancers and play critical roles in tumor development and progression. Furthermore, it has been reported that many lncRNAs regulate gene expression by recruiting chromatin remodeling complexes to specific genomic loci or by controlling transcriptional or posttranscriptional processes. Here we show that an lncRNA termed UPAT [ubiquitin-like plant homeodomain (PHD) and really interesting new gene (RING) finger domain-containing protein 1 (UHRF1) Protein Associated Transcript] is required for the survival and tumorigenicity of colorectal cancer cells. UPAT interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its ß-transducin repeat-containing protein (TrCP)-mediated ubiquitination. Furthermore, we demonstrate that UHRF1 up-regulates Stearoyl-CoA desaturase 1 and Sprouty 4, which are required for the survival of colon tumor cells. Our study provides evidence for an lncRNA that regulates protein ubiquitination and degradation and thereby plays a critical role in the survival and tumorigenicity of tumor cells. Our results suggest that UPAT and UHRF1 may be promising molecular targets for the therapy of colon cancer.

Structural and functional analyses of calcium ion response factors in the mantle of Pinctada fucata.

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis.

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.

Crystal defects induced by chitin and chitinolytic enzymes in the prismatic layer of Pinctada fucata.

Biomineralization, in which organisms create biogenic hard tissues, with hardness or flexibility enhanced by organic-inorganic interaction is an interesting and attractive focus for application of biomimetic functional materials. Calcites in the prismatic layer of Pinctada fucata are tougher than abiotic calcites due to small crystal defects. However, the molecular mechanism of the defect formation remains unclear. Here, chitin and two chitinolytic enzymes, chitinase and chitobiase, were identified as organic matrices related to for the formation of small crystal defects in the prismatic layer. Experiments with a chitinase inhibitor in vivo showed chitinase is necessary to form the prismatic layer. Analysis of calcite crystals, which were synthesized in a chitin hydrogel treated with chitinolytic enzymes, by electron microscopy and X-ray diffraction showed that crystal defects became larger as chitin was more degraded. These results suggest that interactions between chitin and calcium carbonate increase as chitin is thinner.