Hsien-Lu Huang

Phostensin caps to the pointed end of actin filaments and modulates actin dynamics.

Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit encoded by KIAA1949, consists of 165 amino acids and is located between HLA-C and HLA-E gene clusters on human chromosome 6. In this current study, we characterized the biochemical functions of phostensin. Actin dynamics assays using gelsolin-actin seeds showed that phostensin decreases the elongation and depolymerization rates of actin filament pointed ends. The feature of phostensin that binds to the pointed ends of actin filaments was observed through fluorescent single filament binding assay. Taken together, our results suggested that phostensin is an actin filament pointed end-capping protein that is capable of modulating actin dynamics.

FLJ23654 encodes a heart protein phosphatase 1-binding protein (Hepp1).

In this report, we identified the novel protein heart protein phosphatase 1-binding protein (Hepp1), encoded by FLJ23654. Hepp1 associated with protein phosphatase 1 (PP1) by yeast two-hybrid, GST pull-down, co-immunoprecipitation, and far Western blotting assays. Northern blot analysis revealed that Hepp1 mRNA was only expressed in human heart and testis. Recombinant Hepp1 slightly enhanced the enzymatic activity of PP1 and antagonized the ability of phospho-inhibitor-1 or inhibitor-2 to inhibit PP1. Hepp1 protein in human heart tissues was detected by Western blot analysis. Together, our data suggest that Hepp1 can play a role in cardiac functions by working in concert with PP1.

Targeted delivery of an antigenic peptide to the endoplasmic reticulum: application for development of a peptide therapy for ankylosing spondylitis.

The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His6-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS.

Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)2 via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)2 formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)2 of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)2 on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)2 on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs.

Identification and characterization of the actin-binding motif of phostensin.

Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1–129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125–165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.

Immunolocalization of Phostensin in Lymphatic Cells and Tissues

Phostensin binds to the pointed ends of actin filaments and modulates actin dynamics. The genomic location of phostensin is between the HLA-C and HLA-E gene clusters on human chromosome 6, and the mRNA of this protein is predominantly distributed in the spleen, thymus, and peripheral leukocytes. However, the distribution of phostensin in leukocyte cell populations and the subcellular localization have not yet been determined. In this study, an anti-phostensin monoclonal antibody (PT2) that recognizes residues 89–124 of phostensin was prepared and used to examine the subcellular localization and distribution of phostensin in white blood cell populations and in lymphatic tissues. It was found that phostensin is mainly concentrated at the cell periphery and co-localizes with actin filaments. In addition, phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes as well as in the lymphatic tissues, such as the thymus, lymph nodes, and spleen. Phostensin is expressed in the mature lymphocytes of the thymic medulla but not in the immature lymphocytes of the thymic cortex. Taken together, phostensin is a ubiquitous protein in leukocytes, and it may play an important role in modulating the cellular functions of leukocytes.

Characterization of the Recognition Specificity of BH2, a Monoclonal Antibody Prepared against the HLA-B27 Heavy Chain

BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

Identification of the High Molecular Weight Isoform of Phostensin

Phostensin is encoded by KIAA1949. 5′-RACEanalysis has been used to identify the translation start site of phostensin mRNA, indicating that it encodes 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE. This low-molecular-weight phostensin is present in human peripheral blood mononuclear cells and many leukemic cell lines. Phostensin is a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. In the current study, a high-molecular-weight phostensin is identified by using immunoprecipitationin combination with a proteomic approach. This new species of phostensin is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The low-molecular-weight and high-molecular-weight phostensins were named as phostensin-α and phostensin-β, respectively. Although phostensin-α is the C-terminal region of phostensin-β, it is not degraded from phostensin-β. Phostensin-β is capable of associating with PP1 and actin filaments, and is present in many cell lines.

Characterization of the interactions between inhibitor-1 and recombinant PP1 by NMR spectroscopy

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.

Targeted Delivery of the HLA-B27-Binding Peptide into the Endoplasmic Reticulum Suppresses the IL-23/IL-17 Axis of Immune Cells in Spondylarthritis

Ankylosing spondylitis (AS) is highly associated with the expression of human leukocyte antigen-B27 (HLA-B∗27). HLA-B∗27 heavy chain (B27-HC) has an intrinsic propensity to fold slowly, leading to the accumulation of the misfolded B27-HC in the endoplasmic reticulum (ER) and formation of the HLA-B∗27 HC homodimer, (B27-HC)2, by a disulfide linkage at Cys-67. (B27-HC)2 displayed on the cell surface can act as a ligand of the killer-cell Ig-like receptor (KIR3DL2). (B27-HC)2 binds to KIR3DL2 of NK and Th17 cells and activates both cells, resulting in the activation of the IL-23/IL-17 axis to launch the inflammatory reaction in AS patients. However, activation of the IL-23/IL-17 axis originally derived from the HLA-B∗27 misfolding in the ER needs to be characterized. In this study, we delivered two HLA-B∗27-binding peptides, KRGILTLKY and SRYWAIRTR, into the ER by using a tat-derived peptide (GRKKRRQRRR)-His6-ubiquitin (THU) vehicle. Both peptides are derived from the human actin and nucleoprotein of influenza virus, respectively. Our results demonstrated that targeted delivery of both HLA-B∗27-binding peptides into the ER can promote the HLA-B∗27 folding, decrease the levels of (B27-HC)2, and suppress the activation of the IL-23/IL-17 axis in response to lipopolysaccharide. Our findings can provide a new therapeutic strategy in AS.