Cheryl Collins

A Built-in Arginine Finger Triggers the Self-stimulatory GTPase-activating Activity of Rho Family GTPases

Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state. Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an “arginine finger” in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulatedCDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation.

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42 D118Amutant and the lethal, gain of functioncdc42 G12V mutant were examined. Cdc42D118A,C188Sp and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42 D118A phenotype and supported a Cdc24p sequestration model for the mechanism ofcdc42 D118A action. Five cdc42mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42D118A,C188Sp-Cdc24p interactions and could suppress the cdc42 D118A mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. Thecdc42 G12V mutant phenotypes were suppressed by the intragenic T35A and K183–187Q mutations and in skm1Δ and cla4Δ cells but not ste20Δ cells, suggesting that the mechanism of cdc42 G12Vaction is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors ofcdc42 G12V were also identified that displayed a dominant negative phenotype at 16 °C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.

Lyme Arthritis Synovial γδ T Cells Instruct Dendritic Cells via Fas Ligand

γδ T cells participate in the innate immune response to a variety of infectious microorganisms. They also link to the adaptive immune response through their induction of maturation of dendritic cells (DC) during the early phase of an immune response when the frequency of Ag-specific T cells is very low. We observe that in the presence of Borrelia burgdorferi, synovial Vδ1 T cells from Lyme arthritis synovial fluid potently induce maturation of DC, including production of IL-12, and increased surface expression of CD40 and CD86. The activated DC are then able to stimulate the Vδ1 T cells to up-regulate CD25. Both of these processes are initiated primarily by Fas stimulation rather than CD40 activation of DC via high expression of Fas ligand by the Vδ1 T cells. DC are resistant to Fas-induced death due to expression of high levels of the Fas inhibitor c-FLIP. This effect serves to divert Fas-mediated signals from the caspase cascade to the ERK MAPK and NF-κB pathways. The findings affirm the importance of the interaction of certain T cell populations with DC during the early phases of the innate immune response. They also underscore the view that as levels of c-FLIP increase, Fas signaling can be diverted from induction of apoptosis to pathways leading to cell effector function.

Activation of γδ T Cells by Borrelia burgdorferi Is Indirect via a TLR- and Caspase-Dependent Pathway

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are γδ T cells. γδ T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human γδ T cells of the Vδ1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine γδ T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of γδ T cells, are both caspase-dependent.

Squamous cell carcinoma of the cervix: HPV 16 and DNA ploidy as predictors of survival.

In this study, the hypothesis that DNA ploidy and the presence of HPV 16 and HPV 18 DNA affects survival of patients with squamous cell carcinoma of the cervix was tested. Archival paraffin blocks from biopsy and surgical specimens were obtained from 127 women diagnosed in 1977-1984. Determination of DNA ploidy was by flow cytometry and HPV 16 and HPV 18 DNA status by polymerase chain reaction with subsequent dot-blot hybridization. For each patient, age, stage, treatment modality, and 5-year survival were correlated with ploidy and HPV status. HPV 16 DNA was present in 53% of the tumors. HPV 18 was not detected in this population. HPV 16 DNA was found twice as often in Stages IB and IIA than in advanced-stage disease (III and IV). These advanced-stage tumors were more commonly aneuploid. Neither HPV status nor DNA ploidy were predictive of survival for any stage of disease or therapeutic modality.

Increased Caspase Activity Primes Human Lyme Arthritis Synovial γδ T cells for Proliferation and Death

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function and manifesting cytolytic activity. Their numbers often increase during infections, such as human immunodeficiency virus, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we observed that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared with αβ T cells because of increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and because signaling is more efficient through T cell receptor (TCR)-γδ than through TCR-αβ, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than in comparable αβ T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model in which human γδ T cells evolved to function quickly and transiently in an innate fashion.

An Unusual Case Using Dna Polymorphisms to Determine Parentage of Human Remains

The 2-year-old daughter of two farm laborers was reported missing while the farm owner was harvesting corn. Unidentifiable tissues and body parts were subsequently found admixed with silage. Samples of blood collected from the parents of the missing child as well as portions of the tissue recovered from the silage were subjected to analysis of DNA polymorphisms with probes usually used to identify paternity. In addition, allele-specific oligonucleotides were used to detect DNA polymorphism at the DQ alpha locus following DNA amplification using the polymerase chain reaction. In this case, the DNA results established that the tissue recovered from the silage was of human origin and confirmed the probable parentage of the two farm laborers.

Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress.