Biology subjects

Bazan, J. F.

Publications and source records attributed to Bazan, J. F..

5 recordsLinked to original sources

bioRxiv2025

Design principles of a membrane-spanning ubiquitin ligase

Receptor-type E3 ubiquitin ligases are membrane-spanning assemblies that enable extracellular signals to directly control ubiquitylation in the cytoplasm. Despite playing widespread roles in tissue patterning and homeostasis, metabolism, and immunity, their structures and mechanisms remain poorly understood. Using cryo-electron microscopy, integrated with biophysical and functional studies, we visualized an E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex regulates left-right patterning of the body axis and the development of multiple organs, partly by attenuating signaling through the Hedgehog pathway. We find that the MMM complex functions like a fishing pole: a long, flexible helix attached to a membrane platform suspends an activated and precisely oriented RING domain--like a fishhook--to ubiquitylate the cytoplasmic surfaces of target receptors. Our structure explains how mutations in MEGF8 cause multi-organ birth defects in humans and defines a paradigm for receptor regulation by ubiquitylation.

biochemistry↗

bioRxiv2025

Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Iden-tify Covalent TLCD1 Inhibitors

Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds ( stereoprobes) offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.

biochemistry↗

bioRxiv2024

Reanalysis of cryo-EM data reveals ALK-cytokine assemblies with distinct stoichiometries

Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 play important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography (De Munck et al. Nature, 2021) contrasted the 2:2 ALK-ALKAL2 complexes determined by cryo-EM (Reshetnyak et al., Nature, 2021) and X-ray crystallography (Li et al., Nature, 2021). Here, we show based on reanalysis of the cryo-EM data deposited in EMPIAR- 10930 that over half of the ALK-ALKAL2 particles in the dataset are classified into 2D classes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations that resulted in cryo-EM maps displaying strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artefacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 [A] resolution from EMPIAR-10930. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry reconciles a common receptor dimerization mode for ALK and LTK and provides direct evidence for the presence of an ALK-ALKAL2 complex with 2:1 stoichiometry next to the reported 2:2 stoichiometric assembly in the EMPIAR-10930 dataset. Finally, our analysis emphasizes the importance of public deposition of raw cryo-EM data to allow reanalysis and interpretation.

molecular biology↗

bioRxiv2022

TMEM164 is an acyltransferase that forms ferroptotic polyunsaturated ether phospholipids

Ferroptosis is an iron-dependent form of cell death driven by the oxidation of polyunsaturated (PUFA) phospholipids. Large-scale genetic screens have pointed to a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Our understanding of the enzymes involved in PUFA ePL production, however, remains incomplete. Here we show using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions, and targeted lipidomics that the uncharacterized transmembrane protein TMEM164 - genetic ablation of which has been shown to protect cells from ferroptosis - is a cysteine active-center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to furnish PUFA-ePLs. TMEM164-null cells show substantial reductions in PUFA-ePLs, but not PUFA ester phospholipids, supporting that the selective suppression of PUFA-ePLs is sufficient to protect cells from ferroptosis and designating TMEM164 as a key enzyme specifically responsible for regulating this class of lipids.

biochemistry↗

bioRxiv2021

Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength

Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.

developmental biology↗