Biology subjects

Fish, A.

Publications and source records attributed to Fish, A..

8 recordsLinked to original sources

bioRxiv2026

SMIM1 drives an alternative iron uptake pathway

Iron is essential for numerous biological processes, and its abundance is critical in human cells to prevent anemia or toxic overload. Although transferrin receptor-mediated iron uptake is the canonical route, human cells also possess an unstudied alternative pathway. To reveal this missing mechanism, we developed a large-scale, gain-of-function approach to assign genes to a catalogue of quantitative cellular traits. Applying this genetic strategy, we identified SMIM1, the antigen responsible for the rare Vel blood group and a transmembrane protein with unknown function, as a key regulator of iron homeostasis. We found that SMIM1 expression completely suppresses the iron deficiency caused by transferrin receptor loss. Mechanistically, SMIM1 mediates iron uptake through clathrin-dependent endocytosis of hemoglobin (Hb). SMIM1 directly binds Hb via its extracellular HKCK motif, leading to its transport to lysosomes and subsequent iron release. In the human population, genetic variants in SMIM1 influence Hb levels and erythrocyte traits. SMIM1-deficient mice display iron deficiency in early erythroid precursors in response to erythropoietic stress, underscoring the physiological relevance of this process.

cell biology↗

bioRxiv2026

So similar, yet so different: the paradigm of PARP9 macro domain paralogs

Human PARP9 harbours two tandem macro domains, MD1 and MD2, with distinct roles in ADP-ribosylation signaling. Whereas MD1 is a MacroD-type de-MARylase ("eraser"), MD2 functions as a MacroH2A-like "reader" lacking detectable hydrolase activity. How two domains so similar in sequence and fold achieve such divergent functionality has remained unclear. Our de-MARylation assays confirmed this division of labor, even though crystal structures revealed nearly identical /{beta}/ folds and similar binding pockets, and solution NMR shows broadly comparable dynamics. A remarkable distinction, however, emerges from isothermal titration calorimetry, showing that both domains bind free ADP-ribose with comparable affinity (KD = 5.4 and 8.4 M), but through markedly different thermodynamics: MD1 binding is enthalpy-driven and offset by a larger entropic penalty, whereas MD2 binds with weaker enthalpy and a smaller entropic cost. Our ADPr-bound crystal structures rationalized this showing that the distal ribose is positioned differently in the two pockets, connecting to a glycine-rich catalytic loop and a conserved aromatic residue present in active macro domains like MD1, but altered in MD2, while the catalytic asparagine itself is structurally conserved (Asn140/Asn339). Moreover, we show that a single amino acid substitution in MD2 leads to detectable RNA de-MARylation activity without altering its fold. Together, our findings show that an eraser-versus-reader distinction between the macrodomains in PARP9 is encoded in the dynamics of ligand binding and the exact position the distal ribose, revealing an unexpected catalytic plasticity relevant to PARP9s roles in immunity and cancer.

biochemistry↗

bioRxiv2025

Identification of allo- or orthosteric VHH/single-domain antibodies that enhance or block pathogen binding to Siglec-1 on dendritic cells

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/695420v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@e85819org.highwire.dtl.DTLVardef@1eff6b4org.highwire.dtl.DTLVardef@12daa92org.highwire.dtl.DTLVardef@11959e5_HPS_FORMAT_FIGEXP M_FIG C_FIG BackgroundSiglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni. However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. ResultsHere we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1+ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis-binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans-interactions with ligands. ConclusionIn conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.

immunology↗

bioRxiv2024

USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that K48-linked chains are also formed on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about deubiquitination of chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA isopeptide bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis reveals that its mechanistic preference is maintained in evolution. Its kinetic mechanism results in relative enrichment of monoubiquitinated PCNA that could initially promote TLS over HR-like bypass. Taken together, these results suggest that USP1/UAF1 could be important in temporary protection of PCNA against K48- and K63-linked polyubiquitination and highlight this DUB as a potential regulator of DDT pathway choice.

biochemistry↗

bioRxiv2023

Surprising variety in the USP deubiquitinase catalytic mechanism

The USP family of deubiquitinases (DUBs) controls many ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site was so far primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine and a third residue (first critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (second critical residue) is directly adjacent to this first critical residue. Here we study the roles of these critical residues in a subset of USPs and reveal a remarkable variety in function. While USP7 relies on the first critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40 and USP48. Instead, their second critical residue is vital for catalysis. Interestingly, without their respective vital residue USP7, USP15 and USP40 can still perform nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. The unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/550302v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f3fdbeorg.highwire.dtl.DTLVardef@197f836org.highwire.dtl.DTLVardef@28b588org.highwire.dtl.DTLVardef@a62a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisO_LIThe roles of the highly conserved critical residues in USP active sites are poorly understood. Here we show that these two residues have varying importance for catalysis between different USPs. C_LIO_LIExcept for USP7, the majority of USPs does not rely on the canonical third catalytic residue (first critical residue). Instead, the USPs tested rely primarily on the highly conserved second critical residue. C_LIO_LIIn some USPs, either critical residues can accommodate nucleophilic attack (USP7, USP40, USP15). USP1 and USP48 are unable to perform the nucleophilic attack without the second critical residue. C_LI

biochemistry↗

bioRxiv2018

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the \"pocket\" and the \"tunnel\". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

biochemistry↗

bioRxiv2018

Characterisation and structure determination of a llama-derived nanobody targeting the J-base binding protein 1

The J-base Binding Protein 1 (JBP1) contributes to biosynthesis and maintenance of base J ({beta}-D-glucosyl-hydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance (SPR) screening identified Nb6 as a strong binder, recognising JBP1 with a 1:1 stoichiometry and high affinity (kD=30nM). Crystallisation trials of JBP1 in complex with Nb6, yielded crystals diffracting to 1.47[A] resolution.\n\nHowever, the asymmetric unit dimensions and molecular replacement with a nanobody structure, clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in spacegroup P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64[A]. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.\n\nSynopsisA camelid single domain antibody fragment (nanobody) is shown to have high affinity towards its recognition target, the J-base binding protein 1 (JBP1). The serendipitous crystallisation of this nanobody alone, and its crystal structure solution and refinement to 1.64[A] resolution are described. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.

biochemistry↗

bioRxiv2018

Dynamic Kinetochore Size Regulation Promotes Microtubule Capture And Chromosome Biorientation In Mitosis

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. An outer layer of the kinetochore known as the fibrous corona transiently expands in early mitosis and disassembles upon microtubule capture. Neither the functional importance nor the mechanistic basis for this are known. Here we show that the dynein adaptor Spindly and the RZZ kinetochore complex drive fibrous corona formation in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ:Spindly complexes into a corona-like meshwork in cells and in vitro. Concurrent with corona expansion, Spindly potentiates corona shedding by recruiting dynein via three conserved short linear motifs. Expanded, non-sheddable fibrous coronas engage in extensive, long-lived lateral microtubule interactions that persist to metaphase and result in fused sister kinetochores, formation of merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

cell biology↗