A biochemical approach to define the interactome for calpain2 in endothelial cells

Current repositories for protein-protein interactions and high throughput screening methods focus on individual gene products and do not consider the significance of calcium induced conformational changes. These limitations suggest the need for alternative strategies to better define the calpain2 interactome. Affinity capture coupled with LC-MS/MS and proteomic analysis of the recovered proteins provides a powerful approach to identify protein-protein interactions for the heterodimeric calpain2. CAPN2 (rat) was modified to be catalytically incompetent (C105A) and fused with a C-terminal 15 residue peptide optimized for biotinylation by the biotin protein ligase, BirA. The resulting CAPN2*, heterodimerized with truncated CAPNS1, was purified from E. coli, and biotinylated in vitro. Biotinylated calpain2* served as ‘bait’ for streptavidin affinity capture of calpain2 and its interacting proteins from lysates of bovine aortic (BAEC) and human umbilical vein (HUVEC) endothelial cells (ECs). Protein-calpain2 complexes were formed in the presence of calcium to allow EGTA elution of interacting proteins and LC-MS/MS analysis in the absence of an abundance of bait peptides. Capture of the well characterized calpain inhibitor protein calpastatin (CAST), and a known substrate, vimentin provide proof of concept and validates the conformational integrity of the bait calpain2*. Significant overlap between datasets (two from BAEC and one HUVEC) is also encouraging. Of numerous other proteins including several annexins, ANXA1 was confirmed as a substrate for calpain2. Findings are expected to contribute to continuing efforts in the field to better characterize calpain2’s selection of substrates and may reveal other important clues to calpain’s localization and regulation

The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases. © 1999 Cancer Research Campaig

Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale

Mitochondrial dysfunction and biogenesis: do ICU patients die from mitochondrial failure?

Mitochondrial functions include production of energy, activation of programmed cell death, and a number of cell specific tasks, e.g., cell signaling, control of Ca2+ metabolism, and synthesis of a number of important biomolecules. As proper mitochondrial function is critical for normal performance and survival of cells, mitochondrial dysfunction often leads to pathological conditions resulting in various human diseases. Recently mitochondrial dysfunction has been linked to multiple organ failure (MOF) often leading to the death of critical care patients. However, there are two main reasons why this insight did not generate an adequate resonance in clinical settings. First, most data regarding mitochondrial dysfunction in organs susceptible to failure in critical care diseases (liver, kidney, heart, lung, intestine, brain) were collected using animal models. Second, there is no clear therapeutic strategy how acquired mitochondrial dysfunction can be improved. Only the benefit of such therapies will confirm the critical role of mitochondrial dysfunction in clinical settings. Here we summarized data on mitochondrial dysfunction obtained in diverse experimental systems, which are related to conditions seen in intensive care unit (ICU) patients. Particular attention is given to mechanisms that cause cell death and organ dysfunction and to prospective therapeutic strategies, directed to recover mitochondrial function. Collectively the data discussed in this review suggest that appropriate diagnosis and specific treatment of mitochondrial dysfunction in ICU patients may significantly improve the clinical outcome

Lean Phenotype Induced by Methionine Restriction in Mice is Associated with Proteomic Changes in Perivascular Adipose Tissue

Perivascular adipose tissue (PVAT) surrounds blood vessels, is thermogenic, and secretes adipokines that can be vasoprotective. During aging and obesity, PVAT loses its thermogenic capacity, shows increased inflammation, and secretes proatherogenic cytokines. Our collaborative research has previously shown that dietary methionine restriction (MetR) in mice can decrease adiposity, improve glucose sensitivity, and induce PVAT to become more thermogenic. To identify initiating signals from PVAT that correspond to thermogenic conversion, we challenged male C57BL/6 mice with a high fat diet (HFD, 60 kcal% fat, 0.86% methionine) until they weighed \u3e45g (~12 weeks of age). Weight matched mice were then either maintained on this diet or switched to a high fat, methionine restricted diet (60 kcal% fat, 0.12% methionine). We collected PVAT after 3, 5, or 10 days, and analyzed tissues by mass spectrometry using Sequential Window Acquisition of all Theoretical Spectra (SWATH) to identify proteomic signatures. When compared to control, PVAT from methionine restricted conditions exhibited significant differences (p\u3c0.05) in 624, 313, and 502 proteins on days 3, 5, and 10 respectively. Overall, we observed a significant increase in proteins involved in mitochondrial network formation, such as GRP75, a heat-shock protein that our previous data indicate may be a potentially novel thermogenic marker in PVAT. Our study identifies unique protein signatures that are responsive to dietary methionine concentration and are associated with a lean phenotype, despite a HFD. These profiles can be used to identify pathways that may initiate the thermogenic phenotype in PVAT, which is predicted to suppress cardiovascular disease

Inhibition of ovarian tumor growth by targeting the HU177 cryptic collagen epitope.

Evidence suggests that stromal cells play critical roles in tumor growth. Uncovering new mechanisms that control stromal cell behavior and their accumulation within tumors may lead to development of more effective treatments. We provide evidence that the HU177 cryptic collagen epitope is selectively generated within human ovarian carcinomas and this collagen epitope plays a role in SKOV-3 ovarian tumor growth in vivo. The ability of the HU177 epitope to regulate SKOV-3 tumor growth depends in part on its ability to modulate stromal cell behavior because targeting this epitope inhibited angiogenesis and, surprisingly, the accumulation of α-smooth muscle actin-expressing stromal cells. Integrin α10β1 can serve as a receptor for the HU177 epitope in α-smooth muscle actin-expressing stromal cells and subsequently regulates Erk-dependent migration. These findings are consistent with a mechanism by which the generation of the HU177 collagen epitope provides a previously unrecognized α10β1 ligand that selectively governs angiogenesis and the accumulation of stromal cells, which in turn secrete protumorigenic factors that contribute to ovarian tumor growth. Our findings provide a new mechanistic understanding into the roles by which the HU177 epitope regulates ovarian tumor growth and provide new insight into the clinical results from a phase 1 human clinical study of the monoclonal antibody D93/TRC093 in patients with advanced malignant tumors

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models Of Hereditary Hemorrhagic Telangiectasia.

Rationale: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all three HHT genes are components of signal transduction of TGF-β family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. Objective: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression for HHT. Methods and Results: We generated a novel mouse allele (ROSA26Alk1) in which HA-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-overexpression (OE) using the ROSA26Alk1 allele could suppress the development of AVMs in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent overexpression of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 on suppression of phosphor-AKT levels in these ECs. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. Conclusions: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 overexpression or activation can be an effective therapeutic strategy for HHT1 and HHT2 in Alk1- and Eng-inducible knockout (iKO) mice. Further research is required to study whether this therapy could be translated into treatment for humans