Biology subjects

Kikuchi, R.

Publications and source records attributed to Kikuchi, R..

7 recordsLinked to original sources

bioRxiv2024

The splicing factor kinase SRPK1 is a therapeutic target for Peripheral Vascular Disease

IntroductionIn peripheral arterial disease (PAD) anti-angiogenic VEGF-A165b isoform overexpression in monocytes contributes to impaired collateralisation. Serine-arginine protein-kinase-1 (SRPK1) regulates VEGF splicing. To determine whether SRPK1 controlled monocytic VEGF, impairing collateralisation, we investigated SRPK1 inhibition and monocyte-specific knockout in mouse models of and in human monocytes from PAD. MethodsVEGF-A165b activity was measured by co-culture of PAD patients monocytes with endothelial cells with SRPK1 inhibition. Mice with impaired revascularisation due to soluble-frizzled-related-protein-5 knockout (Sfrp5-/-), monocyte-specific Wnt5a gain-of-function (LysM-Wnt5aGOF), or obese mice on a high-fat high-sucrose (HF/HS) diet were subjected to femoral artery ligation and treated with SRPK1 inhibitor. We generated an SRPK1 conditional knockout and crossed it with monocyte-specific (LysM-Cre) driver line to specifically knockout SRPK1 in monocyte lineages. Blood flow was measured by Laser Speckle Imaging before, and for 28 days after surgery. ResultsMonocytes from PAD patients significantly inhibited endothelial cell migration, which was reversed by an anti-VEGF-A165b antibody. Surprisingly, migration was stimulated by SRPK1 inhibition, switching splicing from VEGF-A165b to VEGF-A165a. In Sfrp5-/-, LysM-Wnt5aGOF and HF/HS mouse models of PAD, blood flow was improved by SRPK1 inhibition. Impaired revascularisation in LysM-Wnt5aGOF mice was rescued in LysM-Wnt5aGOF:SRPK1MoKO mice, which had a phenotypic shift towards M2 macrophages. Impaired blood flow recovery was also rescued in obese-SRPK1MoKO mice. ConclusionVEGF splicing in monocytes is regulated differently from VEGF splicing in epithelial or cancer cells suggesting that control of splicing is dependent on cell type and/or environment. SRPK1 inhibition enhances collateralisation in mice, and in human in vitro models of monocyte-dependent impaired angiogenesis. New and NoteworthyA novel potential treatment for peripheral arterial disease (PAD) is described. Inhibition of SRPK1, or knockout in monocytes, induces angiogenesis by preventing splicing to anti-angiogenic VEGF (VEGF-A165b) in patients and animal models. In PAD, monocyte splicing control is different from other cell types and SRPK1 inhibition by drug like compounds can alter macrophage phenotype and reverse PAD in mice using a new cell specific SRPK1-LoxP mouse.

physiology↗

bioRxiv2023

Optineurin provides a mitophagy contact site for TBK1 activation

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase involved in many intracellular processes including innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating autophagy adaptors critical in selective autophagic removal of damaged mitochondria (mitophagy). However, the mechanism by which TBK1 is activated by PINK1/Parkin-mediated mitophagy remains largely unknown. Here, we show that the autophagy adaptor OPTN provides a unique platform for TBK1 activation. The OPTN-ubiquitin and OPTN-autophagy machinery interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact site between damaged mitochondria and the autophagosome formation site. This assembly point serves as a positive feedback loop for TBK1 activation by accelerating hetero-autophosphorylation of the protein. Furthermore, expression of monobodies engineered in this study against OPTN impaired assembly of OPTN at the contact sites as well as the subsequent activation of TBK1 and mitochondrial degradation. Taken together the findings reveal that a positive reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.

cell biology↗

bioRxiv2023

Conserved cis-acting motifs and localization of transcripts and proteins of MFT2 in barley and rice

MFT is an important regulator of seed dormancy in flowering plants. A natural mutation in the A-box motif in the promoter of wheat (Triticum aestivum) MFT2 on chromosome 3A (TaMFT2-3A) has been used to prevent pre-harvest sprouting (PHS) in wheat cultivars in East Asia. Previous research using in situ hybridization showed that TaMFT2-3A is primarily expressed in the seed scutellum. In this study, we analyzed the localization of transcripts and encoded proteins for the TaMFT2 homologs from barley (Hordeum vulgare) and rice (Oryza sativa), HvMFT2 and OsMFT2, respectively. RNA in situ hybridization showed that, like the wheat genes, the rice and barley homologs are primarily expressed in the scutellum, indicating that these three MFT2 genes have a common expression pattern during seed development. Analysis of the cis-acting regulatory elements of their promoter sequences showed that the three MFT2 promoters share eight seed-specific cis-acting RY motifs, which are binding sites for B3-domain transcription factors of the AFL-B3 and VIVIPAROUS1/ABI3-LIKE (VAL) families. In addition, we detected tandemly repeated and partially overlapping A-box motifs in the promoters of HvMFT2, TaMFT2-3B, and TaMFT2-3D, possibly explaining why the natural allele of TaMFT2-3A has been employed in breeding. We generated transgenic rice plants expressing nuclear-localized green fluorescent protein (NLS-2xGFP) and OsMFT2-GFP under the control of a 3-kb fragment of the OsMFT2 promoter. Immunohistochemical staining using anti-GFP antibodies mainly detected GFP in the scutellum and scutellar epithelium, which is an important tissue for initiating germination upon seed hydration. We confirmed these results by confocal microscopy of GFP fluorescence in seeds. Our results suggest that MFT2 expression in rice, barley, and wheat might be regulated by a similar network of transcription factors through multi-RY motifs in the MFT2 promoters, with possible roles in scutellar epithelium development.

plant biology↗

bioRxiv2022

Development of a new 3D tracking system for multiple marmosets under free-moving conditions

Assessment of social interactions and behavioral changes in nonhuman primates is useful for understanding brain function changes during life events and pathogenesis of neurological diseases. The common marmoset (Callithrix jacchus), that lives in a nuclear family like humans, is a useful model, but long-term automated behavioral observation of multiple animals has not been achieved. Here, we developed a Full Monitoring and Animal Identification (FulMAI) system for long-term detection of three-dimensional (3D) trajectories of each individual in multiple marmosets under free-moving conditions by combining video tracking, Light Detection And Ranging, and deep learning. Using this system, identification of each animal was more than 97% accurate. Location preferences and inter-individual distance could be calculated, and deep learning could detect grooming behavior. The FulMAI system allows us to analyze the natural behavior of individuals in a family over their lifetime and understand how behavior changes due to life events together with other data.

animal behavior and cognition↗

bioRxiv2021

Asymmetric Structure of the Native Rhodobacter sphaeroides Dimeric LH1-RC Complex

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Commun. 12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 [A] resolution and from an LH1-RC monomer lacking protein-U ({Delta}U) at 2.64 [A] resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 {beta}-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The {Delta}U LH1-RC monomer revealed a half-moon-shaped structure containing 11 - and 10 {beta}-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of {beta}-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

biochemistry↗

bioRxiv2021

A Novel Membrane Protein in the Rhodobacter sphaeroides LH1-RC Photocomplex

We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 [A] resolution. The LH1 complex forms a C-shaped structure composed of 14 {beta}-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

biochemistry↗

bioRxiv2020

Importin α2 associates with chromatin via a novel DNA binding domain

Nuclear transport of proteins is important for facilitating appropriate nuclear functions. The proteins of the importin family play key roles in nuclear transport as transport receptors for a huge number of nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these non-transport functions of importin are not yet fully understood, especially their molecular-level mechanisms for functioning with chromatin and their consequences. Here, we report the novel molecular characteristics of importin involving binding to diverse sequences in chromatin. We newly identified and characterized a DNA-binding domain--the Nucleic Acid Associating Trolley pole domain (NAAT domain)--in the N-terminal region of importin within the conventional importin {beta} binding (IBB) domain, which was shown to be necessary for nuclear transport of cargo proteins. We propose a stroll and locate model to explain the association of importin with chromatin. This is the first study to delineate the interaction between importin and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin N-terminal region for nuclear transport and chromatin association.

molecular biology↗