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Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in 'immune hot' tumors but not in 'immune cold' tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials., Introduction With encouraging activity in clinical trials that involve the lysosomal inhibitor hydroxychloroquine (HCQ) (1), the identification of palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of chloroquine derivatives (2), [...]

The etiology of severe hemolytic anemia in most patients with recessive hereditary spherocytosis (rHS) and the related disorder hereditary pyropoikilocytosis (HPP) is unknown. Whole-exome sequencing of DNA from probands of 24 rHS or HPP kindreds identified numerous mutations in erythrocyte membrane [alpha]- spectrin (SPTA1). Twenty-eight mutations were novel, with null alleles frequently found in trans to missense mutations. No mutations were identified in a third of SPTA1 alleles (17/48). WGS revealed linkage disequilibrium between the common rHS- linked [[alpha].sup.BH] polymorphism and a rare intron 30 variant in all 17 mutation-negative alleles. In vitro minigene studies and in vivo splicing analyses revealed the intron 30 variant changes a weak alternate branch point (BP) to a strong BP. This change leads to increased utilization of an alternate 3' splice acceptor site, perturbing normal [alpha]-spectrin mRNA splicing and creating an elongated mRNA transcript. In vivo mRNA stability studies revealed the newly created termination codon in the elongated transcript activates nonsense-mediated decay leading to spectrin deficiency. These results demonstrate that a unique mechanism of human genetic disease contributes to the etiology of a third of rHS cases, facilitating diagnosis and treatment of severe anemia and identifying a new target for therapeutic manipulation., Introduction Hereditary spherocytosis (HS), the most common inherited hemolytic anemia in Northern Europeans, affects people of every ethnic background worldwide. A quarter of HS cases exhibit recessive inheritance (rHS) (1-5). [...]

The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARIDlA-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations., A major discovery of recent cancer genome-wide sequencing studies has been the identification of significant alterations in genes responsible for modifying chromatin structure (1). ARID1A, which encodes a component of [...]

Author(s): Mohamed-Ali Hakimi [1]; Daniel A. Bochar [1]; John A. Schmiesing [2]; Yuanshu Dong [1]; Orr G. Barak [1]; David W. Speicher [1]; Kyoko Yokomori [2]; Ramin Shiekhattar (corresponding author) [...]

Research has been conducted on the human erythrocytes spectrin. The use of the sedimentation equilibrium and isothermal titration calorimetry analysis in evaluating the contributions of the terminal nonhomologous domains to spectrin heterodimerization is described.

Research has been conducted on the boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine. The synthesis of this analogue has been carried out demonstrating that it is a slow-binding competitive inhibitor of the binuclear manganese metalloenzyme arginase.

Author(s): Amanpreet Kaur; Marie R. Webster; Katie Marchbank; Reeti Behera; Abibatou Ndoye; Curtis H. Kugel; Vanessa M. Dang; Jessica Appleton; Michael P. OConnell; Phil Cheng; Alexander A. Valiga; Rachel Morissette; [...]

The conformational stability of the human spectrin alpha1 single-motif recombinant peptide was analyzed by utilizing multiple physical methods. The triple-helical alpha1 motif of the 526 kDa alpha/beta heterodimer was characterized as a highly stable independently folding monomeric species. A single homologous segment of spectrin can also fold into a highly stable conformation with a number of physical properties that closely reflected the behavior of the larger spectrin heterodimer.

Properties of the Ca2+-independent barbed-end actin filament capping protein (capZ) were studied by isolating the protein from human polymorphonuclear leukocytes and testing for its actin polymerization inhibitory activity. Capping activity of the capZ was affected by increases in KCl concentrations, as well as by phosphatidylinositol 4,5-bisphosphate. The protein also showed an inability to facilitate nucleation of the actin assembly, sequestering of the acting monomers or severing actin filaments.

The entire primary structure of human erythroid dematin whose sequence includes a homologue of the 'headpiece' sequence discovered at the C terminus of villin has been identified. The headpiece is necessary for villin's stimulation of actin redistribution and microvillar growth. The widely evident expression of dematin transcripts in human tissues indicates that dematin and its homologues may replace villin in villin-regulative tissues to control actin realignment by a phosphorylation-controlled process.

The properties of human red cell spectrin tetramer head-to-head formation were studied using two complementary univalent peptides, the alphaI domain and beta monomers. Tetramers from alphaI and beta monomers bind more easily as temperature decreased from 37 to zero degree centigrade. The interconversion between dimer and tetramer is influenced by the first alpha-beta head-to-head association and dissociation. Removal of the first site on a tetramer increases the rate of association and frees the dimer-tetramer interconversion from temperature constraints.

Isothermal titration calorimetry is used to deduce the mechanism and energetics of the two heterodimer assembly phases. On the basis of this analysis of heterodimer thermodynamics, a detailed model of spectrin dimmer assembly is proposed.

Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the lg domains of a prototypical lg superfamily CAM that contains intradomain disulfide bonds. The lg domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin lg-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five lg domains of Mel-CAM (melanoma CAM)--a protein that is overexpressed in metastatic melanomas--under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's lg-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell-cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's lg domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions. AFM

Cyclosporin A, a cyclic undecapeptide of fungal origin is a potent immunosuppressant with low myelotoxicity (1). It is used to prevent rejection of kidney and liver transplants (2) and in [...]

Amino Acid Sequence Similarity Between Rabies Virus Glycoprotein and Snake Venom Curaremimetic Neurotoxins Rabies virus (RV) is an enveloped, negative-strand RNA virus belonging to the rhabdovirus family, Lyssavirus genus. The [...]