PKC ε Phosphorylates and Mediates the Cell Membrane Localization of RhoA.

Protein kinase C ε (PKC ε ) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKC ε and RhoA was defined. Phosphopeptide mapping revealed that PKC ε phosphorylates RhoA at T127 and S188. Recombinant PKC ε bound to recombinant RhoA in the absence of ATP indicating that the association between PKC ε and RhoA does not require an active ATP-docked PKC ε conformation. Activation of PKC ε resulted in a dramatic coordinated translocation of PKC ε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKC ε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKC ε -RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKC ε is able to recruit RhoA to the cell membrane indicating that the association between PKC ε and RhoA is proximal to the active catalytic site and perhaps independent of a PKC ε -RhoA phosphorylation event. This work demonstrates, for the first time, that PKC ε phosphorylates and modulates the cell membrane translocation of RhoA.

Design and application of a data-independent precursor and product ion repository.

The functional design and application of a data-independent LC-MS precursor and product ion repository for protein identification, quantification, and validation is conceptually described. The ion repository was constructed from the sequence search results of a broad range of discovery experiments investigating various tissue types of two closely related mammalian species. The relative high degree of similarity in protein complement, ion detection, and peptide and protein identification allows for the analysis of normalized precursor and product ion intensity values, as well as standardized retention times, creating a multidimensional/orthogonal queryable, qualitative, and quantitative space. Peptide ion map selection for identification and quantification is primarily based on replication and limited variation. The information is stored in a relational database and is used to create peptide- and protein-specific fragment ion maps that can be queried in a targeted fashion against the raw or time aligned ion detections. These queries can be conducted either individually or as groups, where the latter affords pathway and molecular machinery analysis of the protein complement. The presented results also suggest that peptide ionization and fragmentation efficiencies are highly conserved between experiments and practically independent of the analyzed biological sample when using similar instrumentation. Moreover, the data illustrate only minor variation in ionization efficiency with amino acid sequence substitutions occurring between species. Finally, the data and the presented results illustrate how LC-MS performance metrics can be extracted and utilized to ensure optimal performance of the employed analytical workflows.

Citrulline: a potential immunomodulator in sepsis.

BACKGROUND: Sepsis leads to a complex systemic response of cytokines (both pro- and anti-inflammatory) and more recently recognized adipokine mediators. Endothelial nitric oxide (NO) may be a key component in regulating this response, but the pharmacologic manipulation of endothelial NO via L-arginine supplementation or inhibitors has provided inconsistent clinical data related to outcomes. These failures are related to the metabolism of L-arginine in the liver, toxicity of L-arginine, and asymmetric dimethylarginine inhibition, all of which may explain the "arginine paradox." L-citrulline (CIT) offers a potentially valuable means of supplementing arginine and therefore impacting favorably NO availability. The goal of this study was to determine whether CIT supplementation altered the systemic response of mediators and cytokines in a rat model of sepsis with varying degrees of severity. METHODS: Sepsis was induced with 2 models of cecal ligation and puncture (CLP) of varying severity in Wistar rats. CIT supplementation was provided to half the animals as 8% CIT-supplemented feed for 3 weeks. Baseline mediator levels were assessed in the Wistar rats followed by comparison of the following groups at days 0, 1, and 3: sham-operated; CLP 8-mm (localized); and CLP 12-mm (extensive). The following analyses were performed in the groups: interleukin-6 (IL-6), IL-8, IL-10, resistin, and adiponectin levels (enzyme-linked immunosorbent assay performed in duplicate). L-arginine and CIT were measured with high-performance liquid chromatography combined with mass spectrometry. RESULTS: Ninety-eight Wistar rats were evaluated, and survival was similar in both sepsis models with and without CIT. Serum IL-6 levels were lower in the CIT/CLP 8-mm group compared to the standard rat chow (STD)/CLP 8-mm group (41 vs 117 pg/mL; P = .011) on postoperative day 3. Serum IL-8 and IL-10 responses were similar across all groups. Serum resistin levels were lower in the CIT/CLP 12-mm group compared to the STD/CLP 12-mm group in the more severe sepsis model on day 3 (19 vs 38 ng/mL; P < .0001). The levels of serum L-arginine were greater in the CIT-supplemented animals compared to STD rodent diet animals before surgical insult (86.3 vs 294.0 µM; P = .004). Adiponectin was not affected by CIT supplementation. CONCLUSION: CIT may decrease the proinflammatory mediator response (IL-6 and resistin) without impairing the secretion of anti-inflammatory mediators (IL-10 and adiponectin) and thereby provide a safe means of immunomodulation that preserves the anti-inflammatory mediator response.

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion.

Geldanamycin and its derivative 17AAG [17-(Allylamino)-17-demethoxygeldanamycin, telatinib] bind selectively to the Hsp90 chaperone protein and inhibit its function. We discovered that these drugs associate with mitochondria, specifically to the mitochondrial membrane voltage-dependent anion channel (VDAC) via a hydrophobic interaction that is independent of HSP90. In vitro, 17AAG functions as a Ca(2+) mitochondrial regulator similar to benzoquinone-ubiquinones like Ub0. All of these compounds increase intracellular Ca(2+) and diminish the plasma membrane cationic current, inhibiting urokinase activity and cell invasion. In contrast, the HSP90 inhibitor radicicol, lacking a bezoquinone moiety, has no measurable effect on cationic current and is less effective in influencing intercellular Ca(2+) concentration. We conclude that some of the effects of 17-AAG and other ansamycins are due to their effects on VDAC and that this may play a role in their clinical activity.

Molecular interactions of MMP-13 C-terminal domain with chondrocyte proteins.

Matrix metalloproteinases (MMP)-13 activity is necessary for normal skeletal development and plays a central role in cartilage degeneration associated with osteoarthritis (OA). The studies we described here examine the interactions of the hemopexin domain of MMP-13 with proteins secreted by human chondrocytes in culture. The hemopexin domain of the MMPs and many other proteins in which this structure is found mediates protein function by forming the primary site of interaction with other proteins. We have modified a tandem affinity expression tag (hTAP) to enable efficient expression of the tagged bait protein. In this case the MMP-13 C-terminal domain (CTD) comprises hinge and hemopexin domain, and we immobilized the fusion construct on a column of agarose bound immunoglobin G. The MMP-13 CTD affinity column so generated enabled the efficient and gentle isolation of interacting proteins from the culture medium of human articular chondrocytes. TIMP1 and alpha2-macroglobulin previously shown to interact with MMP-13 as well as several proteins, fibronectin, type VI collagen and xylosyltransferase 1 and several proteoglycans, decorin, syndecan 4 and serglycin not previously recognized as interacting with MMP-13 were identified by mass spectrometry. The interaction between isolated proteins and MMP-13 CTD was verified by yeast two hybrid analysis. We also demonstrated serglycin expression by chondrocytes for the first time and its co localization with MMP-13 in a cytoplasmic granular morphology. The consequence of these interactions remains to be demonstrated, however; binding to MMP-13 suggests a role in the regulation of cartilage degradation.

Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag.

Like other members of the transforming growth factor beta (TGF-beta) family of growth factors, the biological activity of TGF-beta2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-beta2 secreted by hypertrophic chondrocytes we have used expression of TGF-beta2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-beta2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-beta2. Functional interaction was suggested by the inhibition of TGF-beta2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-beta2 in growth cartilage.

Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.