Biology subjects

Hiraizumi, M.

Publications and source records attributed to Hiraizumi, M..

9 recordsLinked to original sources

bioRxiv2026

Structural mechanism of the type IX Retron-Kva2 anti-phage defense system

Retrons are prokaryotic genetic elements that protect bacterial cells from invading phages. The type IX retron comprises a non-coding RNA, a reverse transcriptase (RT), and dual effectors: a putative HEPN nuclease and a winged helix-turn-helix (WH) protein. Here, we show that the type IX retron system from Klebsiella variicola cleaves host rRNAs and tRNAs upon phage infection, mediating anti-phage defense via an abortive-infection mechanism. Cryo-electron microscopy analysis reveals that the RT, HEPN, and WH proteins, along with multicopy single-stranded DNA (msDNA), form a unique, sheet-like supermolecular complex. Notably, the HEPN active site is encircled by the WH and msDNA within the complex, suppressing the nuclease activity prior to phage infection. A phage-encoded exonuclease cleaves the msDNA, likely releasing the HEPN nuclease to cleave host RNAs and induce growth arrest in infected cells. Overall, these findings highlight the structural and functional diversity of prokaryotic anti-phage defense systems.

biochemistry↗

bioRxiv2026

Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: Class 1 DRTs catalyze untemplated synthesis, whereas Class 2 DRTs polymerize noncoding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when Class 1 and Class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5'-ACACAC-3' RNA template to synthesize poly-(dTdG) repeats, whereas DRT3b synthesizes poly-(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for defense.

microbiology↗

bioRxiv2025

Structure-guided development of a potent human B0AT1 inhibitor effective in a mouse model of phenylketonuria

B0AT1 is a neutral amino acid transporter responsible for the (re)absorption in the intestine and kidney. Here, we developed small-molecule inhibitors of B0AT1 as a therapeutic strategy to a transient pocket, located [~]17 [A] away from the substrate-binding site and unique to the outward-open conformation, and stabilize an outward-occluded conformation that prevents the conformational transitions required for transport. Guided by structural insights, we optimized an initial inhibitor (Cinromide) to improve potency and cross-species activity, yielding compound 3, which inhibits both human and mouse B0AT1 with submicromolar IC_50 values. In a PKU mouse model (Pahenu2), oral administration of compound 3 increased urinary Phe excretion and significantly reduced plasma Phe. Our findings identify a druggable allosteric site in B0AT1, demonstrate its utility for achieving potent and selective inhibition in vivo, and establish allosteric blockade as a therapeutic approach for PKU and other SLC6-family transporters.

pharmacology and toxicology↗

bioRxiv2025

Megabase-scale human genome rearrangement with programmable bridge recombinases

Bridge recombinases are a class of naturally occurring RNA-guided DNA recombinases. We previously demonstrated they can programmably insert, excise, and invert DNA in vitro and in bacteria. Here, we report the discovery and engineering of IS622, a simple two-component system capable of universal DNA rearrangements of the human genome. We define strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the IS622 bridge RNA and deep mutational scanning of its recombinase, we achieve up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrate intra-chromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Finally, we provide proof-of-concept for excision of a gene regulatory region or expanded repeats relevant for the treatment of genetic diseases.

bioengineering↗

bioRxiv2024

Rapid protein evolution by few-shot learning with a protein language model

Directed evolution of proteins is critical for applications in basic biological research, therapeutics, diagnostics, and sustainability. However, directed evolution methods are labor intensive, cannot efficiently optimize over multiple protein properties, and are often trapped by local maxima. In silico-directed evolution methods incorporating protein language models (PLMs) have the potential to accelerate this engineering process, but current approaches fail to generalize across diverse protein families. We introduce EVOLVEpro, a few-shot active learning framework to rapidly improve protein activity using a combination of PLMs and protein activity predictors, achieving improved activity with as few as four rounds of evolution. EVOLVEpro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with up to 100-fold improvement of desired properties. We showcase EVOLVEpro for five proteins across three applications: T7 RNA polymerase for RNA production, a miniature CRISPR nuclease, a prime editor, and an integrase for genome editing, and a monoclonal antibody for epitope binding. These results demonstrate the advantages of few-shot active learning with small amounts of experimental data over zero-shot predictions. EVOLVEpro paves the way for broader applications of AI-guided protein engineering in biology and medicine.

bioengineering↗

bioRxiv2024

Bridge RNAs direct modular and programmable recombination of target and donor DNA

Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions, or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes involved in fundamental DNA repair processes such as homologous recombination or in the transposition of foreign genetic material by viruses and mobile genetic elements (MGEs). We report that IS110 insertion sequences, a family of minimal and autonomous MGEs, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables DNA insertion into genomic target sites as well as programmable DNA excision and inversion. The IS110 bridge system expands the diversity of nucleic acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements required for genome design.

genetics↗

bioRxiv2023

Structural insights into the mechanism of the human SGLT2-MAP17 glucose transporter

Selective sodium-glucose cotransporter 2 (SGLT2) plays an important role in glucose reabsorption. SGLT2 inhibitors suppress glucose reabsorption from the kidneys, thus reducing blood glucose levels in type 2 diabetes patients. We and other groups have developed several SGLT2 inhibitors starting from a natural product, phlorizin, but their action mechanisms remain unknown. Here, we elucidated the physiological hSGLT2-MAP17 complex structures bound to five SGLT2 inhibitors using single-particle cryo-electron microscopy. Canagliflozin, dapagliflozin, TA-1887, and sotagliflozin were bound in the outward-facing structure, whereas phlorizin was bound in the inward-open structure. The phlorizin-hSGLT2 interaction biochemically exhibited biphasic binding. Phlorizin weakly binds, via the phloretin motif, from its intracellular side near the Na+-binding site, while strongly interacts from its extracellular side. Unexpectedly, bound Na+ stabilizes the outward-open conformation, while its release allows the transporter to adopt inward-open state. Our results first visualized the Na+-binding and inward-open conformation of hSGLT2-MAP17, clarifying the unprecedented Na+-dependent sugar transport mechanism with MAP17 acting as a scaffold, and may pave the way for development of next-generation SGLT inhibitors.

molecular biology↗

bioRxiv2020

Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated ATP-release channels involved in neural functions including neurotransmission in gustation. Here we present the cryo-EM structures of killifish CALHM1, human CALHM2, and C. elegans CLHM-1 at resolutions of 2.66, 3.51, and 3.60 [A], respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state, and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonomer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the inter-subunit interactions at the transmembrane domain define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels. One Sentence SummaryCryo-EM structures reveal the ATP conduction and oligomeric assembly mechanisms of CALHM channels.

molecular biology↗

bioRxiv2019

Cryo-EM structures capturing the entire transport cycle of the P4-ATPase flippase

In eukaryotic membranes, P4-ATPases mediate the translocation of phospholipids from the outer to inner leaflet and maintain lipid asymmetry, which is critical for protein trafficking and signaling pathways. Here we report the cryo-EM structures of six distinct intermediates of the human ATP8A1-CDC50a hetero-complex, at 2.6-3.3 [A] resolutions, revealing the entire lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.\n\nOne Sentence SummaryCryo-EM reveals lipid translocation by P4-type flippase.

biochemistry↗