Homozygous Apolipoprotein L1 (APOL1) variants G1 and G2 cause APOL1-mediated kidney disease, purportedly acting as surface cation channels in podocytes. APOL1-G0 exhibits various single nucleotide polymorphisms, most commonly haplotype E150K, M228I and R255K ("KIK"; the Reference Sequence is "EMR"), whereas variants G1 and G2 are mostly found in a single "African" haplotype background ("EIK"). Several labs reported cytotoxicity with risk variants G1 and G2 in KIK or EIK background haplotypes, but used HEK-293 cells and did not verify equal surface expression.
View Article and Find Full Text PDFDiabetic retinopathy (DR) is a common complication of diabetes. Approximately 20% of DR patients have diabetic macular edema (DME) characterized by fluid leakage into the retina. There is a genetic component to DR and DME risk, but few replicable loci.
View Article and Find Full Text PDFEngineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome.
View Article and Find Full Text PDFApolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2.
View Article and Find Full Text PDFBackground: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from variant overexpression implicate different subcellular compartments.
View Article and Find Full Text PDFBackground: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of , G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism.
View Article and Find Full Text PDFIgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-dependent manner and is the receptor responsible for the long half-life of IgG.
View Article and Find Full Text PDFDisulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells.
View Article and Find Full Text PDFA cell-based assay employing Madin-Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and β2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans.
View Article and Find Full Text PDFAntibody-drug conjugates (ADC) use monoclonal antibodies (mAb) as vehicles to deliver potent cytotoxic drugs selectively to tumor cells expressing the target. Molecular imaging with zirconium-89 (Zr)-labeled mAbs recapitulates similar targeting biology and might help predict the efficacy of these ADCs. An anti-mesothelin antibody (AMA, MMOT0530A) was used to make comparisons between its efficacy as an ADC and its tumor uptake as measured by Zr immunoPET imaging.
View Article and Find Full Text PDFDMOT4039A, a humanized anti-mesothelin mAb conjugated to the antimitotic agent monomethyl auristatin E (MMAE), was given to patients with pancreatic and ovarian cancer every 3 weeks (0.2-2.8 mg/kg; q3w) or weekly (0.
View Article and Find Full Text PDFThe Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling.
View Article and Find Full Text PDFDelivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable.
View Article and Find Full Text PDFThe genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds.
View Article and Find Full Text PDFMesothelin (MSLN) is an attractive target for antibody-drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine-citrulline linker. The humanized (h7D9.
View Article and Find Full Text PDFCD22 is a cell surface glycoprotein restricted to normal and malignant B-cells and is the target of several anti-CD22 antibody-based cancer therapies. For therapeutic antibody-payload conjugates, it is important to understand the subcellular trafficking of anti-CD22 antibodies to optimize antibody and/or linker-drug properties to maximize antitumor efficacy. It is agreed that anti-CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell-type dependent.
View Article and Find Full Text PDFCilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi-localized membrane protein involved in ciliogenesis.
View Article and Find Full Text PDFThe primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia.
View Article and Find Full Text PDFFc receptor-like 5 (FcRL5/FcRH5/IRTA2/CD307) is a surface protein expressed selectively on B cells and plasma cells. We found that FcRL5 was expressed at elevated levels on the surface of plasma cells from the bone marrow of patients diagnosed with multiple myeloma. This prevalence in multiple myeloma and narrow pattern of normal expression indicate that FcRL5 could be a target for antibody-based therapies for multiple myeloma, particularly antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, where limited expression on normal tissues is a key component to their safety.
View Article and Find Full Text PDFThe reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione.
View Article and Find Full Text PDFUbiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated.
View Article and Find Full Text PDFPalmitoylation of the Wnt and Hedgehog proteins is critical for maintaining their physiological functions. To date, there are no reported studies that characterize the cellular distribution of the palmitoylated forms of these proteins. Here, we describe the subcellular localization of palmitoylated Wnt and Sonic Hedgehog by using a highly sensitive and non-radioactive labeling method that utilizes alkynyl palmitic acid.
View Article and Find Full Text PDFNephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling.
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