Deepti Pradhan

TROSPA, an Ixodes scapularis Receptor for Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.

Regulation of survivin function by Hsp90

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin–Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin–Hsp90 complex may provide a rational approach for cancer therapy.

Rad9 Phosphorylation Sites Couple Rad53 to the Saccharomyces cerevisiae DNA Damage Checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.

Antiviral peptides targeting the west nile virus envelope protein.

ABSTRACT West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

The Spectrin-Ankyrin Skeleton Controls CD45 Surface Display and Interleukin-2 Production

With T cell receptor stimulation, intracellular pools of CD45 and spectrin move to the surface. These processes are coupled. In both peripheral lymphocytes and Jurkat T cells, betaI spectrin and ankyrin associate with CD45. In Jurkat T cells, betaI spectrin peptides suppress surface recruitment of CD45 and CD3 and abrogate T cell activation. Other glycoproteins such as CD43 are not altered by the spectrin peptides. Spectrins effects are mediated by ankyrin, which binds directly to the cytoplasmic domain of CD45 (K(d) = 4.3 +/- 3.0 nM). These data reveal a novel and unexpected contribution of the spectrin-ankyrin skeleton to the control of T lymphocyte function.

Peroxidation-induced perturbations of erythrocyte lipid organization

Peroxidation of erythrocyte membrane lipids by hydrogen peroxide perturbs the lipid bilayer and increases phagocytosis by macrophages. This study addresses the underlying mechanism of these processes, and in particular the role of malondialdehyde, a major byproduct of lipid peroxidation. When erythrocytes were treated with hydrogen peroxide or ascorbate/iron to generate malondialdehyde, or with malondialdehyde itself, only those cells treated with hydrogen peroxide showed increased phospholipid spacing and enhanced phagocytosis. This result indicates that the alterations observed are unique to hydrogen peroxide treatment, and that malondialdehyde does not play a role in inducing these changes in surface properties. Comparison of adherence to human umbilical vein endothelial cells and phagocytosis showed that increased phagocytosis was not mirrored by enhanced adherence. This result suggests that two different signals may mediate recognition of erythrocytes by macrophages and by endothelial cells.

Mechanisms for Recognition and Phagocytosis of Apoptotic Lymphocytes by Macrophages

Erythrocytes have an asymmetric distribution of phospholipids across the bilayer of their plasma membranes, maintained by an ATP-dependent aminophospholipid translocase, and dissipated by activation of a non-specific lipid flipsite. Loss of asymmetry provokes recognition by the reticuloendothelial system. In vitro, enhanced phagocytosis of erythrocytes with a symmetric bilayer can be inhibited by artificial lipid vesicles made of phosphatidylserine (PS), indicating that macrophages recognize the PS that appears on the erythrocyte surface upon loss of asymmetry. It is becoming increasingly clear that these same fundamental membrane structure/function relationships established in the erythrocyte paradigm also apply to lymphocytes. All evidence suggests that lymphocytes maintain an asymmetric transbilayer distribution of phospholipids in their plasma membranes, maintained by an aminophospholipid translocase. Asymmetry is lost as part of the program of cell death, by down-regulation of the translocase and activation of the non-specific lipid flipsite, exposing PS on the cell surface. That PS exposure has functional consequences is demonstrated by the ability of artificial lipid vesicles containing PS to inhibit enhanced phagocytosis of apoptotic lymphocytes by macrophages. However, other signals besides PS are also involved in recognition of apoptotic lymphocytes. Studies with other inhibitors indicate that macrophages also utilize integrin-mediated and lectin-like recognition systems, although each is restricted to either unactivated or activated macrophages. These results indicate that although many fundamental features of recognition by the reticuloendothelial system may be analogous in erythrocytes and lymphocytes, the signals for recognition of apoptotic lymphocytes ae more complex and involve multiple recognition systems.

Phosphatidylserine vesicles inhibit phagocytosis of erythrocytes with a symmetric transbilayer distribution of phospholipids.

In the plasma membrane of erythrocytes, the phospholipids are asymmetrically distributed between the two leaflets of the bilayer. Erythrocytes that have lost this asymmetric distribution are more readily phagocytosed in vitro by macrophages. Surface exposure of phosphatidylserine (PS), normally restricted to the inner leaflet of the bilayer, has been suggested as a signal by which macrophages recognize erythrocytes. To test whether lipid-symmetric erythrocytes are recognized by this mechanism, artificial lipid vesicles made of PS were tested for their ability to inhibit phagocytosis. J774 mouse macrophages, which preferentially phagocytose PS vesicles, were incubated with a mixture of lipid-symmetric erythrocytes and vesicles. PS vesicles reduced phagocytosis of lipid-symmetric erythrocytes to the level of lipid-asymmetric erythrocytes, whereas phosphatidylcholine vesicles had only a limited effect. These results support a role for PS in recognition of erythrocytes and lend credence to the more general hypothesis that PS is involved in the recognition of all blood cells by the reticuloendothelial system.

Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

The leukocyte common antigen, CD45, is a critical immune regulator whose activity is modulated by cytoskeletal interactions. Components of the spectrin-ankyrin cytoskeleton have been implicated in the trafficking and signaling of CD45. We have examined the lateral mobility of CD45 in resting and activated T lymphocytes using single-particle tracking and found that the receptor has decreased mobility caused by increased cytoskeletal contacts in activated cells. Experiments with cells that have disrupted βΙ spectrin interactions show decreased cytoskeletal contacts in resting cells and attenuation of receptor immobilization in activated cells. Applying two types of population analyses to single-particle tracking trajectories, we find good agreement between the diffusion coefficients obtained using either a mean squared displacement analysis or a hidden Markov model analysis. Hidden Markov model analysis also reveals the rate of association and dissociation of CD45-cytoskeleton contacts, demonstrating the importance of this analysis for measuring cytoskeleton binding events in live cells. Our findings are consistent with a model in which multiple cytoskeletal contacts, including those with spectrin and ankyrin, participate in the regulation of CD45 lateral mobility. These interactions are a major factor in CD45 immobilization in activated cells. Furthermore, cellular activation leads to CD45 immobilization by reduction of the CD45-cytoskeleton dissociation rate. Short peptides that mimic spectrin repeat domains alter the association rate of CD45 to the cytoskeleton and cause an apparent decrease in dissociation rates. We propose a model for CD45-cytoskeleton interactions and conclude that the spectrin-ankyrin-actin network is an essential determinant of immunoreceptor mobility.