Carrie Maxwell

Early Structural Rearrangements in the Photocycle of an Integral Membrane Sensory Receptor

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinals beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.

Cultivated vaginal microbiomes alter HIV-1 infection and antiretroviral efficacy in colonized epithelial multilayer cultures.

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

Progastrin Peptides Increase the Risk of Developing Colonic Tumors: Impact on Colonic Stem Cells

Preneoplastic lesions (aberrant crypt foci, hyperplastic/dysplastic polyps) are believed to be precursors of sporadic colorectal tumors (adenomas, adenocarcinomas). Aberrant crypt foci and hyperplastic/dysplastic polyps likely originate from abnormal growth of colonic crypts in response to aberrant queues in the microenvironment of colonic crypts. Thus, identifying factors which regulate homeostatic versus aberrant proliferation/apoptosis of colonocytes, especially stem/progenitor cells, may lead to effective preventative/treatment strategies. On the basis of this philosophy, the role of growth factors/peptide hormones potentially available in the circulation/microenvironment of colonic crypts is being examined extensively. Since the time gastrins were discovered as trophic (growth) factors for gastrointestinal cells, the effect of gastrins on the growth of normal/cancer cells has been investigated, leading to many discoveries. Seminal discoveries in the area of gastrins and colon cancer as it relates to molecular pathways associated with formation of colonic tumors are reviewed and the possible impact on diagnostic/preventative/treatment strategies is discussed.

Adenovirus mediated expression “in vivo” of the chemokine receptor CXCR1

A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian “in vivo” system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of 125I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed “in vivo” was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of 125I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by 125I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.

Annexina2 is Increasingly Expressed and Released Into the Serum of Patients Positive for Colonic Growths/Tumors in Relation to Disease Progression: Diagnostic Implications

Background. Overexpression of AnnexinA2 (ANXA2) has been reported in several epithelial cancers, including colorectal cancers (CRC) (Cancer Letters, 2007). Increasing expression of gastrin gene, and hence progastrin (PG), has also been reported in CRCs. We recently discovered that ANXA2 serves as a non-conventional receptor for PG (Oncogene, 2007). We additionally reported that binding of PGwith extracellular, membrane associated, ANXA2 results in endocytosis and co-localization of PG/ANXA2 intracellularly (Gastro, 2010), and that ANXA2 is required for mediating mitogenic and anti-apoptotic effects of PG on colon cancer cells. ANXA2 is normally involved with intracellular trafficking. During the course of our studies we discovered that ANXA2 is also present on outer membranes of CRC cells, bound to a 29KDa transmembrane protein, and also released into the medium. To further understand physiological significance of these unexpected findings, we examined if ANXA2 is present in serum of patients positive for colorectal growth and if ANXA2 co-localizes with PG in tumor sections, in relation to disease status. Methods, Results and Conclusions. Secretion of ANXA2 (10-50ng/106 cells) was confirmed in conditioned medium (CM) of CRC cells using quantitative Western Blot assay using standard curve with rhANXA2; nontransformed cells were negative. Serum from CRC tumor bearing mice were positive for ANXA2, while control mice were negative. Next, serum samples were obtained from consented patients at time of endoscopy or as discarded samples from CRC patients on day of surgery, as per our IRB protocols. Serum from patients, negative for colonic growths (Normal, N) had low levels of ANXA2 (<1ng/ml), while serum from patients with hyperproliferative growths (Hp), Adenomas (Ad) and adenocarcinomas (AdCA) were on an average positive for 42, 80, and 490 ngs/ml, respectively, suggesting elevation of serum ANXA2 in relation to disease status. Paraffin blocks of Hp, Ads, AdCAs and adjoining N mucosa were obtained as discarded specimens from Department of Pathology and stained immunofluorescently for PG/ANXA2. Hp, Ads and AdCAs were increasingly positive for ANXA2 and PG expression compared to N specimens; ~ 10, 25 and 60 % of respective specimens were positive for intracellular co-localization of PG/ANXA2, suggesting presence of functional PG/ANXA2 axis, confirmed by progressive elevation of nuclear β-catenin and stem cell markers (DCAMKL+1, Lgr5) in relation to disease status. It remains to be determined if, 1) presence of co-localized PG/ANXA2 intracellularly represents a prognostic marker for predicting treatment/recurrence, and 2) if serum ANXA2 levels can be used as a non-invasive diagnostic marker for predicting presence of colorectal growths, stage of disease and/or relapse. Supported by NCI grants CA97959 and CA114264 to PS.

Abstract 2378: Detection of circulating tumors cells (CTCs) in mice, using cancer stem cell (CSC) markers and a novel cell surface marker, AnnexinA2

Detecting CTCs in plasma of patients with solid tumors has developed as a novel method for early detection of metastatic growths (Mets) and relapse of epithelial cancers. The current studies were undertaken to further improve sensitivity/specificity of CTC assays, based on recent developments. Accumulating evidence from many laboratories demonstrates that annexinA2 is increasingly translocated to cell-surface of tumor cells (labeled as CS-ANXA2), and is required for metastasis of cancer cells. We discovered that metastatic transformation of cells was associated with a significant increase in % CSCs co-expressing CS-ANXA2. We hypothesized that Circulating-CSCs (labeled as CTSCs) will likely be positive for CSC-Markers and CS-ANXA2, providing a more robust assay for identifying metastatic-CTCs or CTSCs, which may have the highest potential for seeding Mets; this novel possibility was examined in mice. Athymic nude mice were inoculated with either heat inactivated colon-cancer cells (HCT-116) (control, Gp1), or healthy sterile cells:- which were inoculated either subdermally (to grow primary xenografts) (Gp2) or intraspleenically (to grow liver/lung Mets) (Gp3). Mice were euthanized, when distinct sub-dermal tumors were present in Gp2 mice (wk3, post-inoculation) and blood plasma processed for depletion of WBC. Un-depleted samples had ∼ 109 RBC+WBC cells in all mice. Gp1 mice were negative for CTSCs or CTCs positive for CS-ANXA2. Gp2 mice had ∼40, 100 and 200 epithelial cells/ml blood, positive for CSC-Markers: DCAMKL-1, LGR5 and CD44, respectively, suggesting that DCAMKL-1 may be the most selective marker for CTSCs. Gp3 mice had significantly higher numbers of CTSCs/ml blood, in the order of CD44 (∼220), LGR5 (∼160) and DCAMKL-1 (∼60). CTCs, positive for CS-ANXA2, increased from 120 to 200/ml blood in Gp2 versus Gp3 mice, respectively, suggesting that high numbers of CTCs positive for CS-ANXA2 may be an independent selective marker for metastatic disease. Importantly, CTSCs in Gp3 mice, positive for either CD44 or DCAMKL-1 were also positive for CS-ANXA2. Therefore presence of circulating tumor stem cells (CTSCs) (positive for DCAMKL-1/CD44), co-expressing CS-ANXA2, may provide the most accurate approach for predicting presence of Mets or relapse of the cancer disease. Our studies so far, using fresh plasma samples from patients with colorectal growths (adenomas, adenocarcinomas ± Mets), supports this novel hypothesis. This work was supported by NIH grants CA97959 and CA114264 to PS; NASA NNX09AM08G and NNJ04HD83G to RU. CK and SS were equal contributors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2378. doi:1538-7445.AM2012-2378