Fluorescent Analogues of FRH Peptide: Cu(II) Binding and Interactions with ds-DNA/RNA

Four novel peptidoids, derived from the Phe-Arg-His (FRH) peptide motif, were prepared by replacing the histidine heterocycle with triazole and consequent triazole-fluorophore (coumarin) extension and also replacing arginine with less voluminous lysine. So the constructed Phe-Lys-Ala(triazole) (FKA(triazole)) peptidoids bind Cu2+ cations in water with a strong, nanomolar affinity comparable to the parent FRH and its known analogs, demonstrating that triazole can coordinate copper similarly as histidine. Moreover, even short KA(triazole)coumarin showed submicromolar affinity to Cu2+. Only FKA(triazole)coumarin with free amino groups and its shorter analog KA(triazole)coumarin showed strong induced CD spectra upon Cu2+ cation binding. Thus, KA(triazole)coumarin can be considered as the shortest peptidoid sequence with highly sensitive fluorescent and chiral CD response for Cu2+ cation, encouraging further studies with other metal cations. The FKA(triazole) coumarin peptidoids show biorelevant, 10 µM affinity to ds-DNA and ds-RNA, binding within DNA/RNA grooves. Intriguingly, only peptidoid complexes with Cu2+ strongly stabilize ds-DNA and ds-RNA against thermal denaturation, suggesting significant interactions of Cu2+ cation within the DNA/RNA binding site.

Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.

The EB1–Kinesin-14 complex is required for efficient metaphase spindle assembly and kinetochore bi-orientation

Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1–Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1–Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1–Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.

Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the Drosophila fat body

ABSTRACTHox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Drosophila Hox protein Ultrabithorax (Ubx), we identified the presence of an unconventional nuclear export signal (NES) that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. We show that this unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) in vivo and in vitro. We find that this interaction is tightly regulated in the Drosophila fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other Drosophila and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, our results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels.