L-Type Amino Acid Transporter 1 (LAT1) Promotes PMA-Induced Cell Migration through mTORC2 Activation at the Lysosome.

The mTOR signaling pathway integrates signaling inputs from nutrients, including glucose and amino acids, which are precisely regulated by transporters depending on nutrient levels. The L-type amino acid transporter 1 (LAT1) affects the activity of mTORC1 through upstream regulators that sense intracellular amino acid levels. While mTORC1 activation by LAT1 has been thoroughly investigated in cultured cells, the effects of LAT1 expression on the activity of mTORC2 has scarcely been studied. Here, we provide evidence that LAT1 recruits and activates mTORC2 on the lysosome for PMA-induced cell migration. LAT1 is translocated to the lysosomes in cells treated with PMA in a dose- and time-dependent manner. Lysosomal LAT1 interacted with mTORC2 through a direct interaction with Rictor, leading to the lysosomal localization of mTORC2. Furthermore, the depletion of LAT1 reduced PMA-induced cell migration in a wound-healing assay. Consistent with these results, the LAT1 N3KR mutant, which is defective in PMA-induced endocytosis and lysosomal localization, did not induce mTORC2 recruitment to the lysosome, with the activation of mTORC2 determined via Akt phosphorylation or the LAT1-mediated promotion of cell migration. Taken together, lysosomal LAT1 recruits and activates the mTORC2 complex and downstream Akt for PMA-mediated cell migration. These results provide insights into the development of therapeutic drugs targeting the LAT1 amino acid transporter to block metastasis, as well as disease progression in various types of cancer.

Blocking of the IL-33/ST2 Signaling Axis by a Single-Chain Antibody Variable Fragment (scFv) Specific to IL-33 with a Defined Epitope.

Interleukin 33 (IL-33) is an IL-1 family cytokine that plays a central role in immune system by regulating and initiating inflammatory responses. The binding of IL-33 to the suppressor of tumorigenicity 2 (ST2) receptor induces mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) pathways, thereby leading to inflammatory cytokines production in type 2 helper T cells and type 2 innate lymphoid cells. To develop an antibody specific to IL-33 with a defined epitope, we characterized a single-chain antibody variable fragments (scFvs) clone specific to IL-33, C2_2E12, which was selected from a human synthetic library of scFvs using phage display. Affinity (Kd) of C2_2E12 was determined to be 38 nM using enzyme-linked immunosorbent assay. C2_2E12 did not show cross-reactivity toward other interleukin cytokines, including closely related IL-1 family cytokines and unrelated proteins. Mutational scanning analysis revealed that the epitope of IL-33 consisted of residues 149-158 with key residues being L150 and K151 of IL-33. Structural modeling suggested that L150 and K151 residues are important for the interaction of IL-33 with C2_2E12, implicating that C2_2E12 could block the binding of ST2 to IL-33. Pull-down and in-cell assays supported that C2_2E12 can inhibit the IL-33/ST2 signaling axis. These results suggest that the scFv clone characterized here can function as a neutralizing antibody.

Functional impairment of the HIPK2 small ubiquitin-like modifier (SUMO)-interacting motif in acute myeloid leukemia.

Covalent conjugations of the SUMO-1 moiety on a target protein play important roles in the regulation of cellular protein function. SUMO-conjugation of PML is a regulatory step for PML nuclear body (PML-NB) formation, and HIPK2 is SUMO-conjugated and recruited into the PML-NBs. Although HIPK2 mutations (R861W and N951I) were found in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients, little is known about the underlying mechanisms by which HIPK2 mutations are associated with the pathogenesis of leukemia. Here we show that HIPK2 mutants found in AML and MDS patients are defective in SUMO-interacting motif (SIM) function. Due to defective SIM function, the HIPK2 mutants were not modified with SUMO-1, and not recruited to the PML-NBs. However, the HIPK2 mutants can normally bind to and phosphorylate AML1b. Therefore, the HIPK2 mutants can sequestrate the AML1 complex out of the PML-NBs, resulting in the disruption of AML1-mediated activation of target genes for myeloid differentiation. In addition, the differentiation of K562 blast cells was impaired by the expression of the HIPK2 SIM-defective mutants. These results suggest that HIPK2 targeting into the PML-NBs via the SIMs is crucial for HIPK2-mediated induction of myeloid differentiation, and is associated with AML pathogenesis.

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation.

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin-specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation-induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA).

Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.