Olfactomedin III expression contributes to anoikis-resistance in clonal variants of a human lung squamous carcinoma cell line

Three clonal subpopulations of DLKP, a poorly differentiated squamous lung carcinoma cell line, display striking differences in ability to survive in suspension (anoikis resistance). DLKP-SQ is anoikis resistant (7.5% anoikis at 24 h). In contrast, DLKP-M and DLKP-I are sensitive to anoikis (49.2% and 42.6% respectively). DLKP-I shows increased apoptosis consistently over all time points tested while DLKP-M appear to slow down metabolically and perhaps delays onset of anoikis by undergoing autophagy. Expression microarray analysis identified pronounced differential expression of Olfactomedin 3 (OLFM3) between the clones. High expression of OLFM3 was confirmed at the RNA level by qRT-PCR in DLKP-SQ and at the protein level by Western blotting (within the cell and secreted). Little or no OLFM3 was detected in the other two clones (DLKP-M and DLKP-I). Following siRNA knockdown of OLFM3 in DLKP-SQ, anoikis was increased 2.8-fold to 21% which was intermediate between the anoikis levels in DLKP-SQ and DLKP-M or DLKP-I. This knockdown correlated with increased apoptosis in suspension but not in attached culture conditions. Addition of recombinant OLFM3 reduced anoikis in DLKP-I. This is the first instance of OLFM3 being linked with anoikis resistance in a human cancer cell line.     

Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.Regulated adhesion of lymphocytes is required for immune function. The β2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates lymphocyte adhesion to endothelium, antigen-presenting cells, and virally infected target cells (14). These cell-cell adhesions enable lymphocyte trafficking in and out of lymphoid organs, T-cell activation, and cytotoxicity, respectively (2, 34). Thus, the regulation of LFA-1 adhesiveness is central to adaptive immunity.LFA-1 is a bidirectional receptor in that it mediates both outside-in and inside-out signaling (30). Outside-in signaling is analogous to signaling by conventional receptors and is defined as stimulation of intracellular signaling pathways as a consequence of ligation of LFA-1 with any of its extracellular ligands, such as intracellular adhesion molecule 1 (ICAM-1). Inside-out signaling refers to intracellular signaling events that result in a higher-affinity state of the ectodomain of LFA-1 for its cognate ligands. Regulatory events that mediate inside-out signaling converge on the cytoplasmic tails of the LFA-1 α and β chains, which transduce signals to their ectodomains (14). Signaling molecules implicated in inside-out signaling through LFA-1 include talin, Vav1, PKD1, several adaptor proteins (SLP-76, ADAP, and SKAP-55), the Ras family GTPase Rap1, and two of its effectors, RAPL and RIAM (26). How these proteins interact to activate LFA-1 remains poorly understood.Rap1 is a member of the Ras family of GTPases and has been implicated in growth control, protein trafficking, polarity, and cell-cell adhesion (6). The ability of activated Rap1 to promote LFA-1-mediated lymphocyte adhesion is well established (33). The physiologic relevance of this pathway is highlighted by leukocyte adhesion deficiency type III (LAD III), where immunocompromised patients have a congenital defect in GTP loading of Rap1 in leukocytes (24). LFA-1 is a plasma membrane protein, consistent with its role in cell-cell adhesion, which by definition is a cell surface phenomenon. Paradoxically, the bulk of Rap1 is expressed on intracellular vesicles. We have characterized these vesicles as recycling endosomes and have shown that the intracellular pool of Rap1 can be mobilized by exocytosis to augment the expression of Rap1 at the plasma membranes of lymphocytes, leading to increased adhesion (5). We used a fluorescent probe of activated Rap1 in live cells to show that only the pool of Rap1 at the plasma membrane becomes GTP bound upon lymphocyte activation. Thus, it appears that delivery of Rap1 via vesicular transport to the plasma membrane and activation of the GTPase on that compartment are linked. Among the signaling enzymes known to regulate vesicular trafficking is phospholipase D (PLD). Whereas PLD type 2 (PLD2) is expressed at the plasma membranes of lymphocytes, PLD1 is expressed on intracellular vesicles (29). We now show that PLD1 resides on the same vesicles as Rap1, is delivered along with Rap1 to the plasma membranes of stimulated T cells, and is required for Rap1 activation and T-cell adhesion.     

Immune and behavioral consequences of microglial reactivity in the aged brain

Bidirectional communication between the immune system and the brain is essential for mounting the appropriate immunological, physiological, and behavioral responses to immune activation. Aging, however, may impair this important bi-directional interaction. In support of this notion, peripheral infection in the elderly is associated with an increased frequency of behavioral and cognitive complications. Recent findings in animal models of aging and neurodegenerative disease indicate that microglia, innate immune cells of the brain, become primed or reactive. Understanding age- and disease-associated alterations in microglia is important because glia (microglia and astrocytes) play an integral role in propagating inflammatory signals that are initiated in the periphery. In this capacity, brain glia produce inflammatory cytokines that target neuronal substrates and elicit a sickness-behavior syndrome that is normally beneficial to the host organism. Increased reactivity of microglia sets the stage for an exaggerated neuroinflammatory cytokine response following activation of the peripheral innate immune system, which may underlie subsequent long-lasting behavioral and cognitive deficits. In support of this premise, recent findings indicate that stimulation of the peripheral immune system in aged rodents causes exaggerated neuroinflammation that is paralleled by cognitive impairment, prolonged sickness, and depressive-like complications. Therefore, the purpose of this review is to discuss the new evidence that age-associated priming of microglia could play a pathophysiological role in exaggerated behavioral and cognitive sequelae to peripheral infection.     

Design and synthesis of novel pyrimidine hydroxamic acid inhibitors of histone deacetylases

Inhibition of histone deacetylase activity represents a promising new modality in the treatment of a number of cancers. A novel HDAC series demonstrating inhibitory activity in cell proliferation assays is described. Optimisation based on the introduction of basic amine linkers to effect good drug distribution to tumour led to the identification of a compound with oral activity in a human colon cancer xenograft study associated with increased histone H3 acetylation in tumour tissue.     

Deconvolution and Database Search of Complex Tandem Mass Spectra of Intact Proteins: A COMBINATORIAL APPROACH*

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks. This raises both pattern recognition and combinatorial challenges for spectral deconvolution. We present MS-Deconv, a combinatorial algorithm for spectral deconvolution. The algorithm first generates a large set of candidate isotopomer envelopes for a spectrum, then represents the spectrum as a graph, and finally selects its highest scoring subset of envelopes as a heaviest path in the graph. In contrast with other approaches, the algorithm scores sets of envelopes rather than individual envelopes. We demonstrate that MS-Deconv improves on Thrash and Xtract in the number of correctly recovered monoisotopic masses and speed. We applied MS-Deconv to a large set of top-down spectra from Yersinia rohdei (with a still unsequenced genome) and further matched them against the protein database of related and sequenced bacterium Yersinia enterocolitica. MS-Deconv is available at http://proteomics.ucsd.edu/Software.html.Top-down proteomics is a mass spectrometry-based approach for identification of proteins and their post-translational modifications (PTMs)1 (1–14). Unlike the “bottom-up” approach where proteins are first digested into peptides and then a peptide mixture is analyzed by mass spectrometry, the top-down approach analyzes intact proteins. Thus, it has advantages in detecting and localizing PTMs as well as identifying multiple protein species (e.g. proteolytically processed protein species). Despite its advantages, top-down proteomics presents many challenges. These include requirement of high sample quantity, sophisticated instrumentation, protein separation, and robust computational analysis tools. For this reason, top-down proteomics has rarely been used for analyzing complex mixtures (12–18), and it is typically used to study single purified proteins. However, this situation is quickly changing with recent top-down studies of complex protein mixtures (14, 19).Because of the existence of natural isotopes, fragment ions of the same chemical formula and charge state are usually represented by a collection of spectral peaks in tandem mass spectra called an isotopomer envelope. The monoisotopic mass of a chemical formula is the sum of the masses of the atoms using the principal (most abundant) isotope for each element. Spectral deconvolution focuses on grouping spectral peaks into isotopomer envelopes. By doing so, the charge state and monoisotopic mass of each envelope are effectively determined. A complex multi-isotopic peak list in the m/z space is translated into a simple monoisotopic mass list that is easier to analyze.Given the monoisotopic mass and charge state of a fragment ion, its theoretical isotopic distribution can be predicted by assuming the fragment ion has an average elemental composition with respect to its mass (20) or using its precise elemental composition if the protein is known. Exploiting this, many deconvolution methods use theoretical isotopic distributions to detect and evaluate candidate isotopomer envelopes, which is the envelope detection problem (Fig. 1). To evaluate the fit of a candidate envelope to its theoretical isotopic distribution, many metrics have been proposed (20–32).Open in a separate windowFig. 1.Envelope detection. a, a theoretical isotopic distribution is predicted with the monoisotopic mass and charge state of a fragment ion. b, an observed envelope is detected by mapping peaks in the theoretical distribution to the spectrum. c, match between the theoretical isotopic distribution and the observed envelope. d, the theoretical isotopic distribution is scaled (the intensities of the peaks are multiplied by a constant) to have the best fit with the intensities of peaks in the observed envelope. Finally, a score for the observed envelope can be computed by comparing it with the intensity-scaled theoretical isotopic distribution.The candidate envelopes often overlap and share peaks, leading to a combinatorial problem of selecting the list of envelopes that best explains the spectrum (Fig. 2). In contrast to the well studied envelope detection problem, the envelope selection problem remains poorly explored. Most deconvolution algorithms follow a simple greedy approach to selecting the set of envelopes where the highest scoring envelopes are iteratively selected and removed from the spectrum. Although this approach often generates reasonable sets of envelopes for simple spectra, its performance deteriorates in cases of complex spectra.Open in a separate windowFig. 2.Envelope selection problem. Overlapping envelopes lead to a difficult combinatorial problem of selecting an optimal set of envelopes. We illustrate two cases where a deconvolution method that follows a greedy envelope selection outputs the envelope E₂, whereas the optimal solution consists of the envelopes E₁ and E₃. Example a illustrates the case where envelopes do not share peaks, and example b illustrates the case where envelopes share a spectral peak (E₁ and E₃).In particular, the greedy approach performs well when the envelopes are distributed sparsely along the m/z axis. Large proteins have many fragments that appear in multiple charge states. The high number of envelopes/peaks and the small m/z spread of the fragments with high charge states result in narrow m/z regions with high peak density. In these peak-dense regions, envelopes may overlap and share peaks, and the greedy approach and even manual interpretation often fail to find the optimal combination of envelopes (supplemental Fig. 1).Several methods have been proposed to explore the envelope selection problem. McIlwain et al. (33) presented a dynamic programming algorithm for selecting a set of envelopes such that the m/z ranges of the envelopes do not overlap. This non-overlapping condition becomes too restrictive for complex spectra of intact proteins. Samuelsson et al. (34) proposed a method that follows a non-negative sparse regression scheme. Du and Angeletti (35) and Renard et al. (36) addressed the envelope selection problem as a statistical problem of variable selection and used LASSO to solve it.Here, we present MS-Deconv, a combinatorial algorithm for spectral deconvolution. MS-Deconv (i) generates a large set of candidate envelopes, (ii) constructs an envelope graph encoding all envelopes and relationships between them, and (iii) finds a heaviest path in the envelope graph. Although the envelope graph of a complex spectrum is large (exceeding a million nodes in some cases), the heaviest path algorithm can efficiently find an optimal set of envelopes. MS-Deconv explicitly scores combinations of candidate envelopes rather than individual envelopes as in previous approaches.We tested MS-Deconv on a data set of top-down spectra from known proteins and evaluated the monoisotopic masses recovered by MS-Deconv. A mass was classified as a true positive if it was matched to the monoisotopic mass of a theoretical fragment ion of the protein within a specific parts per million (ppm) tolerance. We compared the performance of MS-Deconv with the widely used Thrash (20) and Xtract (37) and demonstrated that, with a few exceptions, MS-Deconv recovers more true positive masses. For example, for the collisionally activated dissociation (CAD) spectrum of bacteriorhodopsin (BR) with charge 10, the percentage of true positive masses among the top 150 masses is above 70% for MS-Deconv and less than 50% for Thrash. Additionally, MS-Deconv is ∼33 times faster than Thrash and 4 times faster than Xtract. Furthermore, MS-Deconv implements some user-friendly features: (i) outputs the set of peptide sequence tags, (ii) provides protein and spectral annotations, and (iii) allows one to inspect the recovered envelopes. We also tested MS-Deconv on a large LC-MS/MS data set from Yersinia rohdei (with a still unsequenced genome) (19). Y. rohdei is a non-pathogenic bacterium that is often used as a simulant for the potential bioterrorism agent Yersinia pestis, the causative agent of plague. We applied MS-Deconv to extract monoisotopic mass lists from top-down spectra and compared the mass lists with those reported by Thrash. We used ProSightPC (38) and the spectral alignment algorithm (39) to identify related proteins from a protein database of Yersinia enterocolitica (with a closely related and sequenced genome). The results demonstrated that MS-Deconv reported more matched fragments than Thrash for most proteins. Additionally, using spectral alignment, we identified eight proteins in Y. rohdei that were not reported in the ProSightPC-based searches (19) of the Y. enterocolitica protein database.