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Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α2β2γ2heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and the identification of a hockey stick-like motif that controls activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and selective substrate binding of a lysosomal hydrolase through its protein-binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx motifs bind to the µ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the µ or β subunits of these adaptors as well as the γ/σ1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the γ/σ1 or α/σ2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the β1/µ1 and β2/µ2 hemicomplexes, and the individual β or µ subunits, was extremely weak or undetectable. The γ/σ1 and α/σ2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position –4 compromised binding to the γ/σ1 hemicomplex, whereas minimally affecting binding to α/σ2. There was an excellent correlation between binding to the α/σ2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxxφ and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxxφ motifs bind to the μ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the μ or β subunits of these adaptors as well as the γ/σ1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the γ/σ1 or α/σ2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the β1/μ1 and β2/μ2 hemicomplexes, and the individual β or μ subunits, was extremely weak or undetectable. The γ/σ1 and α/σ2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position −4 compromised binding to the γ/σ1 hemicomplex, whereas minimally affecting binding to α/σ2. There was an excellent correlation between binding to the α/σ2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

The GGAs (Golgi-localizing,γ-adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitroglutathione S-transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.

The expression of mutants with deletions in the N-terminal signal-anchor sequence of cytochrome P450 2C2 and His-tag fusions was examined in Escherichia coli to determine the influence of N-terminal sequences on expression of the protein. Two mutants predicted to be translocated across the membrane inhibited bacterial growth. In other mutants, deletion of the N-terminal transmembrane domain (residues 2–20) reduced expression of functional P450 by about 75% and further deletion of the following linker sequence (residues 21–27) resulted in a modest further decrease. Expression of the mutant with residues 2–27 deleted contrasts with the lack of expression of functional protein if only the linker was deleted, which suggests that the linker sequence is critical for expression only if the protein is inserted into the membrane by the transmembrane domain. Fusion proteins of green fluorescent protein with full-length P450 2C2 and 2C2(Δ2–20) were predominantly membrane-associated in vivo as determined by fluorescence microscopy. Subcellular fractionation of bacteria expressing these proteins and extraction of the proteins from the membrane by high salt or alkaline buffer demonstrated that P450 2C2 was an integral membrane protein while 2C2(Δ2–20) was a peripheral membrane protein that associated with the membrane mainly by hydrophobic interactions. Residues 1–27 of P450 2C2 fused to green fluorescent protein resulted in a redistribution of fluorescence from cytosol to membrane, which, with the deletion studies, indicates that the P450 signal-anchor is both necessary and sufficient for normal membrane targeting and is the sole transmembrane domain of cytochrome P450 2C2 in bacteria. Addition of a His-tag at the N-terminus completely restored wild-type expression levels to the 2C2(Δ2–20) mutants in bacteria. In insect cells, functional 2C2(Δ2-20) was not expressed but an N-terminal His-tag also restored full expression. The increase in expression may be related to decreased association with the membrane mediated by the His-tag.

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from thetrans-Golgi network (TGN). The β1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the β chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the γ subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the γ hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the γ subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-γ hinge, but not by GST-γ appendage. However, the hinge and appendage domains of γ function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the γ and β1 subunits of AP-1.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) have revolutionized the management of relapsed and refractory B cell malignancies. Despite high initial response rates, many patients with acute lymphoblastic leukemia (ALL) ultimately relapse after CART19. In contrast, most patients with non-Hodgkin lymphoma experience only partial or no responses. Collectively, <50% of patients treated with CART19 achieve durable disease remission. Identification of the biology responsible for these failures is central to improving CAR T cell efficacy. Several clinical reports have demonstrated that a common cause of resistance to CART19 is antigen escape, in which ALL clones emerge that have lost surface expression of CD19. The mechanisms of antigen escape that have been recognized to date all rely on disruption of CD19genomic loci or transcribed CD19 mRNA; alterations of fully-translated CD19 protein that lead to CART19 failure have not been described.

Ruella: AbClon: Consultancy, Research Funding; viTToria biotherapeutics: Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; Tmunity: Patents & Royalties. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Cytochromes P450 (P450) are anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains in P450 2C2 is a glycine-rich region from residues 22 to 28. To examine the role of this region, deletion and substitution mutations were constructed, and the activities and spectral properties were determined for the mutant proteins expressed in COS-1 cells, insect cells, and bacteria. Deletion of residues 22 to 28 or substitution of 7 valines for this region inactivated the proteins in COS-1 cells, and no P450 species was detected for these mutations in bacteria or insect cells. Substitution of the three glycine residues with alanine or proline or the entire sequence from 22 to 28 with 7 alanines did not reduce lauric acid hydroxylase activity of the proteins expressed in COS-1 cells. Reducing the number of alanines substituted to 4, 3, and 2 progressively decreased activity in COS-1 cells to undetectable levels when 2 alanines were substituted. The loss of activity in COS-1 cells correlated with decreased expression of hemoprotein with a reduced difference spectrum of 450 nm (P450 species) and a corresponding increase in the inactive P420 species in insect cells and bacteria. The activities expressed per nanomole of P450 in insect microsomes were similar for P450 2C2 and the alanine substitution mutants, including the mutant with 2 alanines which was inactive in COS-1 cells. The rates of conversion of P450 to P420 resulting from incubation at 48 °Cin vitrowere not changed sufficiently to explain the increase in expressed P420 observed for the mutants with 3 or 7 alanines substituted. These data are consistent with a role for the residue 22–28 region as a linker that facilitates the folding of P450; however, once the protein is properly folded into the functional P450 species, this region has little influence on the stability and activity of the enzyme.

In cytochrome P450 2C2, the region which links the N-terminal signal anchor with the catalytic domain contains a highly conserved proline-rich region with the sequence, 30-PPGPTPFP-37. Mutation of proline-30 or proline-33 diminished activities of the mutants expressed in COS-1 cells (Chen, C., and Kemper, B. (1996)J. Biol. Chem.271, 28697–28611). Substitution of alanine, proline, or arginine for glycine-32 abolished laurate hydroxylase activity of the proteins expressed in COS-1 cells, which suggests that this residue is also functionally important. To determine the basis for the decreased activity in COS-1 cells, the activities and spectral properties of mutant proteins expressed in insect cells and bacteria were determined. Substitution of alanine for either proline-30 or -33 resulted in reduced expression in insect cells of functional cytochrome P450 hemoprotein and an increase in the expression of inactive cytochrome P420. In contrast, substitution of alanine for proline-31, -35, or -37 resulted in hemoproteins with spectra similar to cytochrome P450 2C2 so that the amount of cytochrome P450 expressed in insect cells correlated with the activities of the mutants in COS-1 cells. The laurate hydroxylase activities per nanomole of cytochrome P450 in insect microsomes were similar for wild type and all mutants, indicating that, once folded, the catalytic activity of membrane-bound cytochrome P450 was not affected by the mutations. Expression in bacteria resulted in diminished expression of cytochrome P450 for all mutants, with the greatest decrease for the proline-30 and -33 mutants, and increased cytochrome P420. In contrast to the insect cell studies, the proline-30 and -33 mutants were inactive, while the other mutants had specific activities 30–70% of cytochrome P450 2C2. These data are consistent with a role for the proline-rich region in efficient assembly of cytochrome P450 2C2 in eukaryotic cells. Mutations of this region also may affect the conformational integrity of the proteins, which was revealed by assays of solubilized bacterially expressed proteins.

Background Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes.Results Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs.Conclusions Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.