title: Angiopoietin-like protein 2, a chronic inflammatory mediator, is a new target induced by TGF-β1 through a Smad3-dependent mechanism

authors: Lee, Hee Jae

subjects: Angptl2

abstract: Angiopoietin-like protein 2 (Angptl2) levels are increased by obesity and obesity-related pathological conditions, and it is considered to be an important adipocyte-derived inflammatory mediator. In contrast, the multifunctional cytokine TGF-β1 has been reported to be augmented in obesity of rodents and humans, but inhibits adipocyte differentiation in vitro. Here we demonstrate that TGF-β1 induces expression of the Angptl2 gene through a Smad3-dependent pathway in RAW264.7 macrophage cells, primary peritoneal macrophages, and differentiated 3T3-L1 adipocytes. Transcriptional induction of the Angptl2 gene by TGF-β1 was dependent on the Smad3 protein which binds to the Smad Binding Element (SBE) region located on the Angptl2 promoter. Macrophages with Smad3 knocked down by small interfering RNA showed reduction of TGF-β1-induced Angptl2 expression. These findings may provide insight into the molecular mechanisms of the increased expression of Angptl2 and TGF-β1 in obesity.

text: Angiopoietin-like protein 2, a chronic inflammatory mediator, is a new target induced by TGF-β1 through a Smad3-dependent mechanism Angiopoietin-like protein 2 (Angptl2) levels are increased by obesity and obesity-related pathological conditions, and it is considered to be an important adipocyte-derived inflammatory mediator. In contrast, the multifunctional cytokine TGF-β1 has been reported to be augmented in obesity of rodents and humans, but inhibits adipocyte differentiation in vitro. Here we demonstrate that TGF-β1 induces expression of the Angptl2 gene through a Smad3-dependent pathway in RAW264.7 macrophage cells, primary peritoneal macrophages, and differentiated 3T3-L1 adipocytes. Transcriptional induction of the Angptl2 gene by TGF-β1 was dependent on the Smad3 protein which binds to the Smad Binding Element (SBE) region located on the Angptl2 promoter. Macrophages with Smad3 knocked down by small interfering RNA showed reduction of TGF-β1-induced Angptl2 expression. These findings may provide insight into the molecular mechanisms of the increased expression of Angptl2 and TGF-β1 in obesity.

year: 2013

title: Understanding allosteric enzyme regulation using macromolecular rate theory

authors: Hamill, Carlin James

subjects: Enzyme

abstract: Enzyme catalysed reaction rates produce a curved temperature dependence with a temperature optimum, Topt. Traditionally enzyme temperature dependence has been modelled by the Arrhenius and Eyring-Polanyi equations. Deviations from
these models at elevated temperatures have traditionally been attributed to enzyme denaturation. Increasing evidence shows that enzyme denaturation is insufficient to fully explain the curvature for enzyme catalysed rates with temperature.

Macromolecular rate theory (MMRT) accounts for these deviations from early models by introducing the concept of heat capacity (Cp). During an enzymatic reaction, the heat capacity is reduced as the transition state complex forms (due to enzyme rigidification), resulting in an overall negative change in heat capacity (ΔCp^‡) for the reaction. Heat capacity is a function of the number and energies of the vibrational modes for the individual molecules. Enzyme-ligand binding will influence the energies of the vibrational modes of the molecular complex
therefore affecting an enzymes’ heat capacity and its catalytic rate via ΔCp^‡. Notably, a form of enzyme regulation (called allostery) utilises ligand binding at a site other than the active site to effect changes in catalytic rate.

The underlying mechanisms of allostery are poorly understood. Here, the mechanisms of allostery are explored using a model isomaltase enzyme and MMRT to investigate changes in heat capacity. Several mutants of the model MalL enzyme were designed, produced and purified. These mutations aimed to either
alter the structural integrity or mimic ligand binding in the C-terminal domain. Using a Stopped-Flow apparatus, these mutants were characterised kinetically and using crystallographic techniques, two structures were solved at high resolution.

Targeted mutations approximately 30 Å from the active site showed significant changes in enzyme rate. Overall, the two new structures of MalL were very similar to the apo-enzyme showing only very minor structural deviations. Changes to the rate can thus be attributed to changes in enzyme dynamics and heat capacity.
These results are a promising first step in linking changes in enzymatic activity with changes to enzyme dynamics and heat capacity, using MMRT as a model.

text: Understanding allosteric enzyme regulation using macromolecular rate theory Enzyme catalysed reaction rates produce a curved temperature dependence with a temperature optimum, Topt. Traditionally enzyme temperature dependence has been modelled by the Arrhenius and Eyring-Polanyi equations. Deviations from
these models at elevated temperatures have traditionally been attributed to enzyme denaturation. Increasing evidence shows that enzyme denaturation is insufficient to fully explain the curvature for enzyme catalysed rates with temperature.

Macromolecular rate theory (MMRT) accounts for these deviations from early models by introducing the concept of heat capacity (Cp). During an enzymatic reaction, the heat capacity is reduced as the transition state complex forms (due to enzyme rigidification), resulting in an overall negative change in heat capacity (ΔCp^‡) for the reaction. Heat capacity is a function of the number and energies of the vibrational modes for the individual molecules. Enzyme-ligand binding will influence the energies of the vibrational modes of the molecular complex
therefore affecting an enzymes’ heat capacity and its catalytic rate via ΔCp^‡. Notably, a form of enzyme regulation (called allostery) utilises ligand binding at a site other than the active site to effect changes in catalytic rate.

The underlying mechanisms of allostery are poorly understood. Here, the mechanisms of allostery are explored using a model isomaltase enzyme and MMRT to investigate changes in heat capacity. Several mutants of the model MalL enzyme were designed, produced and purified. These mutations aimed to either
alter the structural integrity or mimic ligand binding in the C-terminal domain. Using a Stopped-Flow apparatus, these mutants were characterised kinetically and using crystallographic techniques, two structures were solved at high resolution.

Targeted mutations approximately 30 Å from the active site showed significant changes in enzyme rate. Overall, the two new structures of MalL were very similar to the apo-enzyme showing only very minor structural deviations. Changes to the rate can thus be attributed to changes in enzyme dynamics and heat capacity.
These results are a promising first step in linking changes in enzymatic activity with changes to enzyme dynamics and heat capacity, using MMRT as a model.

year: 2020

title: Piahana Hou: a Ngati Rahiri rangatira

authors: Hart, Philip

abstract: Piahana Hou, as the half-brother of Karauna Hou, had an illustrious ancestry, but to Pakeha he was known as, briefly, the skipper of a river steamer and, for most of his life, as a labourer. His interests in several blocks of land enabled him to obtain money through selling some of his holdings, but he never became prosperous. He had minimal involvement in mining at Te Aroha.
Piahana came to prominence not as a rangatira but as a drunk, who was even involved in a bar fight at Te Aroha. Despite this weakness, his high status meant that hundreds attended his splendid tangi. Unlike some other rangatira, he did not use his status to play an important role in either Maori or Pakeha society.

text: Piahana Hou: a Ngati Rahiri rangatira Piahana Hou, as the half-brother of Karauna Hou, had an illustrious ancestry, but to Pakeha he was known as, briefly, the skipper of a river steamer and, for most of his life, as a labourer. His interests in several blocks of land enabled him to obtain money through selling some of his holdings, but he never became prosperous. He had minimal involvement in mining at Te Aroha.
Piahana came to prominence not as a rangatira but as a drunk, who was even involved in a bar fight at Te Aroha. Despite this weakness, his high status meant that hundreds attended his splendid tangi. Unlike some other rangatira, he did not use his status to play an important role in either Maori or Pakeha society.

year: 2016