Hsien-Bin Huang

Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2.

Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 ∼1 nm) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6–11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1–8 (I2-(9–204)) or 100–204 (I2-(1–99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9–13 (I2-(14–204)) resulted in a large decrease in inhibitory potency (IC50 ∼800 nm), whereas further COOH-terminal deletion (I2-(1–84)) caused a moderate decrease in inhibitory potency (IC50∼10 nm). Within residues 9–13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6–20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6–38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1–120) but not I2-(85–204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14–84 of inhibitor-2 and residues 6–38 of phospho-DARPP-32 share elements that are important for interaction with PP1.

Anti–citrullinated protein antibodies bind surface-expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA. METHODS ACPAs were purified from pooled ACPA-positive RA sera by cyclic citrullinated peptide-conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor alpha (TNFalpha) production and NF-kappaB DNA binding activity of the cells were measured by enzyme-linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis. RESULTS ACPAs specifically enhanced TNFalpha production and increased the DNA-binding activity of NF-kappaB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface-expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S-transferase-tagged recombinant Grp78 (97.52 kd) became a 72-kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFalpha production. CONCLUSION We clearly demonstrated that ACPAs enhance NF-kappaB activity and TNFalpha production in monocyte/macrophages via binding to surface-expressed citrullinated Grp78.

Protein phosphatase 1 regulation by inhibitors and targeting subunits

Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli. This preparation is limited by several key differences in its properties compared with native PP1. In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, Mr 32,000). Mutations at Y272 in the β12/β13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2. Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased Km for phosphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1. Replacement of residues 274–330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS). More limited alterations in residues in β12, β13, and β14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.

The Microcystins and Nodularins: Cyclic Polypeptide Inhibitors of PP1 and PP2A

The serine/threonine phosphatases are inhibited by a variety of natural toxins, including the microcystins and nodularins. Progress in understanding the details of the biosynthetic origin and the binding of these compounds is discussed, as is the progress made in synthesizing the members of these families. Additionally, the work by several groups to either synthesize simplified analogues that are still potent, or introduce selectivity for PP1 over PP2A are discussed. Finally, the properties of a series of five new truncated analogues are examined.

Decreased microRNA(miR)-145 and increased miR-224 expression in T cells from patients with systemic lupus erythematosus involved in lupus immunopathogenesis

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with abnormal T cell immune responses. We hypothesized that aberrant expression of microRNAs (miRNAs) in T cells may contribute to the pathogenesis of SLE. First, we analysed the expression profiles of 270 human miRNAs in T cells from five SLE patients and five healthy controls and then validated those potentially aberrant‐expressed miRNAs using real‐time polymerase chain reaction (PCR). Then, the expression of mRNAs regulated by these aberrant‐expressed miRNAs was detected using real‐time PCR. Finally, miRNA transfection into Jurkat T cells was conducted for confirming further the biological functions of these miRNAs. The initial analysis indicated that seven miRNAs, including miR‐145, miR‐224, miR‐513‐5p, miR‐150, miR‐516a‐5p, miR‐483‐5p and miR‐629, were found to be potentially abnormally expressed in SLE T cells. After validation, under‐expressed miR‐145 and over‐expressed miR‐224 were noted. We further found that STAT1 mRNA targeted by miR‐145 was over‐expressed and apoptosis inhibitory protein 5 (API5) mRNA targeted by miR‐224 was under‐expressed in SLE T cells. Transfection of Jurkat cells with miR‐145 suppressed STAT1 and miR‐224 transfection suppressed API5 protein expression. Over‐expression of miR‐224 facilitates activation‐induced cell death in Jurkat cells. In the clinical setting, the increased transcript levels of STAT1 were associated significantly with lupus nephritis. In conclusion, we first demonstrated that miR‐145 and miR‐224 were expressed aberrantly in SLE T cells that modulated the protein expression of their target genes, STAT1 and API5, respectively. These miRNA aberrations accelerated T cell activation‐induced cell death by suppressing API5 expression and associated with lupus nephritis by enhancing signal transducer and activator of transcription‐1 (STAT)‐1 expression in patients with SLE.

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Aβ peptides

Aggregated β‐amyloid (Aβ) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a β‐sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Aβ sequence remains unclear. To explore the correlation between Aβ sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full‐length and fragment‐truncated Aβ peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C‐terminus, particularly the amino acids of 17–21, 25–35, and 41–42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17–21, 25–35 or 41–42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22–24 or residues 36–40 had little effect on toxicity and aggregative ability. While the N‐terminal residues 1–16 may not play a major role in neurotoxicity and aggregation, a lack of N‐terminal fragment Aβ peptide, (e.g. Aβ17–35), does not display the neurotoxicity of either full‐length or 17–21, 25–35 truncated Aβ peptides.

Increased miR-223 expression in T cells from patients with rheumatoid arthritis leads to decreased insulin-like growth factor-1-mediated interleukin-10 production

We hypothesized that the aberrant expression of microRNAs (miRNAs) in rheumatoid arthritis (RA) T cells was involved in the pathogenesis of RA. The expression profile of 270 human miRNAs in T cells from the first five RA patients and five controls were analysed by real‐time polymerase chain reaction. Twelve miRNAs exhibited potentially aberrant expression in RA T cells compared to normal T cells. After validation with another 22 RA patients and 19 controls, miR‐223 and miR‐34b were over‐expressed in RA T cells. The expression levels of miR‐223 were correlated positively with the titre of rheumatoid factor (RF) in RA patients. Transfection of Jurkat cells with miR‐223 mimic suppressed insulin‐like growth factor‐1 receptor (IGF‐1R) and transfection with miR‐34b mimic suppressed cAMP response element binding protein (CREB) protein expression by Western blotting. The protein expression of IGF‐1R but not CREB was decreased in RA T cells. The addition of recombinant IGF‐1‐stimulated interleukin (IL)‐10 production by activated normal T cells, but not RA T cells. The transfection of miR‐223 mimic impaired IGF‐1‐mediated IL‐10 production in activated normal T cells. The expression levels of SCD5, targeted by miR‐34b, were decreased in RA T cells after microarray analysis. In conclusion, both miR‐223 and miR‐34b were over‐expressed in RA T cells, but only the miR‐223 expression levels were correlated positively with RF titre in RA patients. Functionally, the increased miR‐223 expression could impair the IGF‐1‐mediated IL‐10 production in activated RA T cells in vivo, which might contribute to the imbalance between proinflammatory and anti‐inflammatory cytokines.

Aberrant expression of microRNAs in T cells from patients with ankylosing spondylitis contributes to the immunopathogenesis

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in AS T cells involved in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analysed by real‐time polymerase chain reaction (PCR). Thirteen miRNAs were found potentially differential expression. After validation, we confirmed that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells and the expression of miR‐221 and let‐7i were correlated positively with the Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine in AS patients. The protein molecules regulated by miR‐16, miR‐221 and let‐7i were measured by Western blotting. We found that the protein levels of Toll‐like receptor‐4 (TLR‐4), a target of let‐7i, in T cells from AS patients were decreased. In addition, the mRNA expression of interferon (IFN)‐γ was elevated in AS T cells. Lipopolysaccharide (LPS), a TLR‐4 agonist, inhibited IFN‐γ secretion by anti‐CD3+anti‐CD28 antibodies‐stimulated normal T cells but not AS T cells. In the transfection studies, we found the increased expression of let‐7i enhanced IFN‐γ production by anti‐CD3+anti‐CD28+ lipopolysaccharide (LPS)‐stimulated normal T cells. In contrast, the decreased expression of let‐7i suppressed IFN‐γ production by anti‐CD3+anti‐CD28+ LPS‐stimulated AS T cells. In conclusion, we found that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells, but only miR‐221 and let‐7i were associated with BASRI of lumbar spine. In the functional studies, the increased let‐7i expression facilitated the T helper type 1 (IFN‐γ) immune response in T cells.

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: rational modifications imparting PP1 selectivity

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.

Structure-function studies on Taiwan cobra long neurotoxin homolog.

A novel long neurotoxin homolog was purified from Naja naja atra (Taiwan cobra) venom using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. The determined protein sequence was essentially the same as that deduced from the cDNA amplified by reverse transcriptase-polymerase chain reaction. The long neurotoxin homolog exhibited an activity that inhibited acetylcholine-induced muscle contractions, as with N. naja atra cobrotoxin. The degree of inhibition caused by the addition of long neurotoxin homolog was approximately 70% of that observed with the addition of cobrotoxin. Unlike the well-known short and long neurotoxins, this neurotoxin homolog contained two additional cysteine residues forming a disulfide linkage in the N-terminal region. Circular dichroism measurement and computer models of the neurotoxin reveal that its secondary structure was not abundant in beta-sheet as noted with short and long neurotoxins. This less ordered structure may be associated with the lower activity noted with the long neurotoxin homolog. Together with the finding that the known long neurotoxin homologs exclusively appear in the venoms of the Naja and Bungarus genera, the long neurotoxin homologs should represent an evolutionary branch from the long and short neurotoxins in the Elapidae family.