Brian Hampton

LRP1 Protects the Vasculature by Regulating Levels of Connective Tissue Growth Factor and HtrA1

Objective—Low-density lipoprotein receptor–related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1−/−) on a low-density lipoprotein receptor–deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. Approach and Results—We found smLRP1−/− mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1−/− vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. Conclusions—Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

The reverse in-gel kinase assay to profile physiological kinase substrates.

Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the α catalytic subunit of protein kinase CK2 (CK2α) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2α and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.

Developmental alcohol exposure leads to a persistent change on astrocyte secretome

Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome in vitro. Animals were treated with ethanol (3.5 g/kg) or saline between postnatal day (P)10–30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids. Twenty‐four hour conditioned media of cells obtained from ethanol‐ or saline‐treated animals (ET‐CM or SAL‐CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here, we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET‐CM to SAL‐CM. Among the 59 proteins that were found to be reduced in ET‐CM we observed components of the extracellular matrix such as laminin subunits α2, α4, β1, β2, and γ1 and the proteoglycans biglycan, heparan sulfate proteoglycan 2, and lumican. Proteins with trophic function such as insulin‐like growth factor binding protein 4, pigment epithelium‐derived factor, and clusterin as well as proteins involved on modulation of proteolysis such as metalloproteinase inhibitor 1 and plasminogen activator inhibitor‐1 were also reduced. In contrast, pro‐synaptogeneic proteins like thrombospondin‐1, hevin as well as the modulator of extracelular matrix expression, angiotensinogen, were found increased in ET‐CM. The analysis of interactome maps through ingenuity pathway analysis demonstrated that the amyloid beta A4 protein precursor, which was found reduced in ET‐CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET‐CM. Taken together our results strongly suggest that early exposure to teratogens such as alcohol may lead to an enduring change in astrocyte secretome.

Regulation of Itch and Nedd4 E3 Ligase Activity and Degradation by LRAD3

Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.

Removal of N‐Terminal Blocking Groups from Proteins

Two enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Curr. Protoc. Protein Sci. 63:11.7.1‐11.7.20.

UNIT 11.7 Removal of N-Terminal Blocking Groups from Proteins

Two enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N‐terminally blocked proteins using acid‐catalyzed hydrolysis or methanolysis, hydrazinolysis, and β‐elimination after acid‐catalyzed N‐O shift. Alternate protocols describe chemical removal of acetyl and longer‐chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.