Biology subjects

Kaminishi, T.

Publications and source records attributed to Kaminishi, T..

8 recordsLinked to original sources

bioRxiv2024

Transcriptional Dynamics Uncover the Role of BNIP3 in Mitophagy during Muscle Remodeling in Drosophila

Differentiated muscle cells contain myofibrils and well-organized organelles, enabling powerful contractions. Muscle cell reorganization occurs in response to various physiological stimuli; however, the mechanisms behind this remodeling remain enigmatic due to the lack of a genetically trackable system. Previously, we reported that a subset of larval muscle cells is remodeled into adult abdominal muscle through an autophagy-dependent mechanism in Drosophila. To unveil the underlying mechanisms of this remodeling, we performed a comparative time-course RNA-seq analysis of isolated muscle cells with or without autophagy. It revealed both transcriptional dynamics independent of autophagy and highlighted the significance of BNIP3-mediated mitophagy in muscle remodeling. Mechanistically, we found that BNIP3 recruits autophagic machinery to mitochondria through its LC3-interacting (LIR) motif and minimal essential region (MER), which interact with Atg8a and Atg18a, respectively. Loss of BNIP3 leads to a substantial accumulation of larval mitochondria, ultimately impairing muscle remodeling. In summary, this study demonstrates that BNIP3-dependent mitophagy is critical for orchestrating the dynamic process of muscle remodeling. Impact statementA time-course RNA-seq analysis of muscle remodeling reveals transcriptional dynamics independent of autophagy and highlights the significance of BNIP3 in mitochondrial degradation in vivo.

cell biology↗

bioRxiv2024

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7[A]) and on empty ribosomes (2.09 [A]). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

microbiology↗

bioRxiv2024

The Rubicon-WIPI axis regulates exosome biogenesis during aging

Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.

cell biology↗

bioRxiv2023

Golgi membrane proteins YIPF3 and YIPF4 regulate turnover of the Golgi apparatus through autophagy

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the autophagosome. Using quantitative proteomic analysis and two Golgiphagy-reporter systems that we developed, we found that five-transmembrane Golgi-resident proteins, YIPF3 and YIPF4, constitute a Golgiphagy receptor. The YIPF3-YIPF4 complex interacts with LC3B, GABARAP, and GABARAPL1 via the LIR motif in YIPF3, whose stability is dependent on YIPF4. Phosphorylation of the LIR in YIPF3 seems to be required for YIPF3-ATG8 interaction. Moreover, expression of the YIPF3 LIR mutant caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays that we established will pave the way for future studies to obtain deeper insights into Golgiphagy.

cell biology↗

bioRxiv2023

Selective plane activation structured illumination microscopy

Resolution enhancement in structured illumination microscopy is hindered when volumetric samples are observed owing to background signals from out-of-focus planes. In this study, we utilized selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. We demonstrated high-resolution fluorescence imaging of volumetric samples, including dense fluorescent objects in a cell spheroid.

biophysics↗

bioRxiv2021

Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

biochemistry↗

bioRxiv2020

A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria employ an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-EM to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S rRNA central to decoding site maturation, and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

microbiology↗

bioRxiv2018

The third-generation tetracycline, KBP-7072, exploits and reveals a new potential of the primary tetracycline binding pocket

Antibiotic resistance is a growing threat to human health requiring the discovery or development of new anti-infectives. As such, KBP-7072 is a novel tetracycline derivative that exhibits broad-spectrum activity against Gram-positive and -negative bacterial strains. To determine the mechanism of action of KBP-7072 and understand how its unique C9 extension can be used to combat the growing problem of antibiotic resistance we determined the structure of KBP-7072 bound to the bacterial 30S ribosomal subunit, the inhibitory target of typical tetracyclines. We show that KBP-7072 binds to the primary tetracycline binding site on the 30S ribosomal subunit consistent with it acting as a protein synthesis inhibitor that blocks A-site occupation. Moreover, the unique chemical nature of KBP-7072{acute}s C9 extension leads to a distinctive interaction pattern with the 30S subunit that distinguishes KBP-7072 from the third-generation tetracycline, Tigecycline, and thus expands the interaction potential of the primary tetracycline binding pocket.

biochemistry↗