Biology subjects

Hintzen, J.

Publications and source records attributed to Hintzen, J..

8 recordsLinked to original sources

bioRxiv2026

Sortase-mediated enrichment of ubiquitinated proteins from complex samples

Proteolytic processing is typically used to reveal post-translational modifications for mass spectrometric analysis. Here, we show that proteolysis of ubiquitinated proteins additionally generates a chemically addressable diglycine remnant that can be enzymatically remodeled. We exploit the ability of sortase to recognize ubiquitin-derived diglycine motifs as nucleophiles and ligate functionalized LPXTG peptides directly onto ubiquitin-remnant peptides. This reaction enables installation of a biotin affinity handle on defined ubiquitinated substrates and peptides derived from mammalian cell lysates, including lysine- and N-terminal ubiquitination. The resulting branched conjugates generate characteristic fragment ions that facilitate their identification. We further develop a bifunctional sortase donor containing an internal trypsin cleavage site, enabling affinity capture followed by conversion of enriched ubiquitin sites into a compact TGG mass-spectrometric signature. Alternatively, reversible sortase chemistry can regenerate the original diglycine-containing peptide. These studies establish ubiquitin-derived proteolytic remnants as latent chemical handles that can be converted into modular affinity and analytical tags, providing an antibody-independent approach to interrogating protein ubiquitination.

biochemistry↗

bioRxiv2026

A mitochondrial program encodes brain vascular reserve

Brain resilience depends on collateral vessels whose geometries preserve blood flow when primary arteries are perturbed. How these protective vascular architectures are developmentally established remains unknown. Using longitudinal in vivo imaging in zebrafish, we show that mitochondrial state in embryonic angiogenic tip cells encodes the topology of basal and surface brain collateral networks. Mechanistically, microRNA-125a establishes the bioenergetic and redox balance of endothelial tip-cell mitochondria through conserved repression of the metabolic regulator PGC1a. Disruption of the microRNA-125a-PGC1a axis uncouples mitochondrial capacity from redox buffering in developing brain tip cells, redirecting their migration toward sparse, incompletely connected collateral network topologies and increasing adult cerebrovascular vulnerability. Consistent with this mechanism, humans with subclinical cerebrovascular injury exhibit reduced circulating microRNA-125a levels associated with incomplete basal collateral configurations. Together, these findings identify mitochondrial state as an instructive and conserved developmental program that encodes brain vascular reserve.

developmental biology↗

bioRxiv2025

Quinazolinone and Phthalazinone Inhibitors of the HDAC6/Ubiquitin Protein-Protein Interaction

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that regulates diverse cytosolic acetylation through its two catalytic deacetylase domains and a C-terminal zinc finger ubiquitin-binding domain (ZnF-UBD). This ZnF-UBD mediates key protein-protein interactions (PPIs) that couple deacetylation and ubiquitin-dependent degradation. While most HDAC6 inhibitors target the catalytic domains, the ZnF-UBD represents an underexplored target. Here, we validate previously reported small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction and describe novel N-alkyl moieties based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone and phthalazinone scaffolds, a literature and modeling-guided scaffold hop revealed potential for an extended phthalazinone series. Results obtained both in fluorescence polarization (FP) and differential scanning fluorimetry (DSF) confirm this hypothesis. Additionally, late-stage diversification yields compounds with improved predicted physicochemical properties. Finally, machine-learning-based co-folding affinity predictions correlate with experimental IC{square}{square} rank order, highlighting their utility in PPI inhibitor design. These studies continue expanding the chemical space of HDAC6 ZnF-UBD inhibitors and build upon existing foundations for future therapeutic and mechanistic exploration of HDAC6- ubiquitin signaling.

biochemistry↗

bioRxiv2024

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and G proteins may serve this role, we performed siRNA screening of G subunits and found that Gi2 and Gq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the G residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.

molecular biology↗

bioRxiv2022

Hematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic hemogenic endothelium

Multipotent hematopoietic stem/progenitor cells (HSPCs) generate all mature blood cells in the erythroid, lymphoid, and myeloid lineages. HSPCs are initially produced in the embryo, via transdifferentiation of hemogenic endothelial cells (hemECs) in the aorta-gonad mesonephros (AGM). HSPCs in the AGM are functionally heterogenous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. This knowledge could inform approaches to overcome the dysregulation of HSPC heterogeneity associated with poor outcomes of autologous transplants. Here we discovered that loss of microRNA (miR)-128 (miR-128{Delta}/{Delta}) in zebrafish leads to an expansion of hemECs forming replicative HSPCs in the AGM, and a skew towards the erythroid and lymphoid lineages in larval and adult stages. Furthermore, we found that inhibiting miR-128 during the differentiation of human pluripotent stem cells into hemECs, but not during the endothelial-to-hematopoietic transition, recapitulated the lineage skewing. In vivo, expression of wild-type miR-128 in endothelium restored the blood lineage distribution in miR-128{Delta}/{Delta} zebrafish. We found that miR-128 represses the expression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b, and thus promotes Wnt and Notch signaling in hemECs. De-repression of cskn1a1 resulted in hemECs generating replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in hemECs forming lymphoid-biased HSPCs in the AGM and relative mature blood cells in adult. We propose that HSPC heterogeneity is established in hemogenic endothelium prior to transdifferentiation and is programmed in part by Wnt and Notch signaling modulation.

developmental biology↗

bioRxiv2021

Substrate Specificity for Human Histidine Methyltransferase SETD3

Histidine methyltransferase SETD3 plays an important role in human biology and diseases. Previously, we showed that SETD3 catalyzes N3-methylation of histidine 73 in {beta}-actin (Kwiatkowski et al., 2018). Here we report integrated synthetic, biocatalytic, biostructural and computational analyses on human SETD3-catalyzed methylation of {beta}-actin peptides possessing histidine and its structurally and chemically diverse mimics. Our enzyme assays supported by biostructural analyses demonstrate that SETD3 has a broader substrate scope beyond histidine, including N-nucleophiles on the aromatic and aliphatic side chains. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free-energy simulations provide insight into binding geometries and the free energy barrier for the enzymatic methyl transfer to histidine mimics, further supporting experimental data that histidine is the superior SETD3 substrate over its analogs. This work demonstrates that human SETD3 has a potential to catalyze efficient methylation of several histidine mimics, overall providing mechanistic, biocatalytic and functional insight into {beta}-actin histidine methylation by SETD3.

biochemistry↗

bioRxiv2020

Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

Endothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single G proteins had little effect but combined depletion of Gi and Gq/11 blocked all known PECAM1-dependent responses. Re-expression of Gi2 and Gq but not Gi1 and Gi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gi2 and Gq/11 (Q306 in Gi1/3), determines participation in flow signaling. We developed pull-down assays for measuring G activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gs, which identified latrophilins (ADGRLs) as specific upstream interactors for Gi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gi2 and Gq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of G proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via G protein-dependent and -independent mechanisms.

molecular biology↗

bioRxiv2019

N-Glycome regulation limits the transdifferentiation of endothelial cells into hematopoietic stem cells

Hematopoietic stem and progenitor cells (HSPCs) that establish and maintain the blood system in adult vertebrates arise from the transdifferentiation of hemogenic endothelial cells (hemECs) during embryogenesis. This endothelial-to-hematopoietic transition (EHT) is tightly regulated, but the mechanisms are poorly understood. Here, we show that microRNA (miR)-223-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. Single cell RNA-sequencing revealed that miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid/myeloid lineages by suppressing the expression of mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in N-linked protein glycosylation. High-throughput glycomics of ECs lacking miR-223 showed a decrease of high mannose versus sialylated complex/hybrid sugars on N-glycoproteins involved in EHT such as the metalloprotease Adam10. Endothelial-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied the aberrant HSPC production of miR-223 mutants. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of their hematopoietic fate. One Sentence SummaryThe N-glycan "sugar code" governs the hematopoietic fate of endothelial cells and regulates blood stem cell production in vivo.

developmental biology↗