Dale Hereld

Fluorescence Resonance Energy Transfer Imaging Reveals that Chemokine-Binding Modulates Heterodimers of CXCR4 and CCR5 Receptors

Background Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. Methodology/Principal Findings Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5STA and CCR5Δ4. Conclusions/Significance Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.

How human leukocytes track down and destroy pathogens: lessons learned from the model organism Dictyostelium discoideum

Human leukocytes, including macrophages and neutrophils, are phagocytic immune cells that capture and engulf pathogens and subsequently destroy them in intracellular vesicles. To accomplish this vital task, these leukocytes utilize two basic cell behaviors—chemotaxis for chasing down infectious pathogens and phagocytosis for destroying them. The molecular mechanisms controlling these behaviors are not well understood for immune cells. Interestingly, a soil amoeba, Dictyostelium discoideum, uses these same behaviors to pursue and injest its bacterial food source and to organize its multi-cellular development. Consequently, studies of this model system have provided and will continue to provide us with mechanistic insights into the chemotaxis and phagocytosis of immune cells. Here, we review recent research in these areas that have been conducted in the Chemotaxis Signal Section of NIAID’s Laboratory of Immunogenetics.

The Safe Passage Study: Design, Methods, Recruitment, and Follow-Up Approach

BACKGROUND The Safe Passage Study is a large, prospective, multidisciplinary study designed to (1) investigate the association between prenatal alcohol exposure, sudden infant death syndrome (SIDS), and stillbirth, and (2) determine the biological basis of the spectrum of phenotypic outcomes from exposure, as modified by environmental and genetic factors that increase the risk of stillbirth, SIDS, and in surviving children, fetal alcohol spectrum disorders. METHODS The results provided are based on an interim assessment of 6004 women enrolled, out of the 12,000 projected, from the Northern Plains, US, and Cape Town, South Africa, areas known to be of high risk for maternal drinking during pregnancy. Research objectives, study design, and descriptive statistics, including consent, recruitment, and retention information, are provided. RESULTS Overall visit compliance is 87%, and includes prenatal, delivery/newborn, and postnatal contacts through 1 year post-delivery. Pregnancy outcome ascertainment is 98% prior to medical chart review; less than 2% of women withdraw. Consent for the use of DNA and placental tissue exceed 94%, and consent to participate in the autopsy portion of the study is 71%. CONCLUSIONS The Safe Passage Study is the first multi-site study of SIDS and stillbirth to integrate prospectively collected exposure information with multidisciplinary biological information in the same maternal and fetal/infant dyad using a common protocol. Essential components of the study design and its success are close ties to the community and rigorous systems and processes to ensure compliance with the study protocol and procedures.

A modified Timeline Followback assessment to capture alcohol exposure in pregnant women: Application in the Safe Passage Study

Prenatal alcohol exposure (PAE) has been linked to poor pregnancy outcomes, yet there is no recognized standard for PAE assessment, and the specific effects of quantity, frequency, and timing remain largely unknown. The Safe Passage Study was designed to investigate the role of PAE in a continuum of poor peri- and postnatal outcomes. The objective of this manuscript is to describe the rationale for, and feasibility of, modifications to the traditional Timeline Followback (TLFB) for collecting PAE information in a large cohort of pregnant women. Participants from the Northern Plains region (in the United States) and Cape Town, South Africa, were followed prospectively using a modified 30-day TLFB interview, administered up to five times, to obtain detailed PAE information. Required modifications for our population included capturing information regarding sharing, type/brand, container size, and duration, in order to accurately record the amount of alcohol consumed. PAE status was defined for 99.9% of the 11,892 enrolled pregnancies at least once during pregnancy and for 92% across all trimesters. Of 53,823 drinks reported, 98% had all items necessary for standard drink computation. Sharing was reported for 74% of drinks in Cape Town, South Africa and for 10% in the Northern Plains. Compared to referent values from the traditional TLFB, 74% and 67% of drinks had different alcohol-by-volume and container size, respectively. Furthermore, a statistically significant difference was found between the number of containers reported and the number of standard drinks computed, using information from the modified TLFB. This is the first study of this size to wholly encompass all of these changes into a single measure in order to more accurately calculate daily consumption and assess patterns over time. The methods used to collect PAE information and create alcohol exposure measures likely increased the accuracy of standard drinks reported and could be generalized to other populations.

Drinking and Smoking Patterns during Pregnancy: Development of Group-based Trajectories in the Safe Passage Study

Precise identification of drinking and smoking patterns during pregnancy is crucial to better understand the risk to the fetus. The purpose of this manuscript is to describe the methodological approach used to define prenatal drinking and smoking trajectories from a large prospective pregnancy cohort, and to describe maternal characteristics associated with different exposure patterns. In the Safe Passage Study, detailed information regarding quantity, frequency, and timing of exposure was self-reported up to four times during pregnancy and at 1 month post-delivery. Exposure trajectories were developed using data from 11,692 pregnancies (9912 women) where pregnancy outcome was known. Women were from three diverse populations: white (23%) and American Indian (17%) in the Northern Plains, US, and mixed ancestry (59%) in South Africa (other/not specified [1%]). Group-based trajectory modeling was used to identify 5 unique drinking trajectories (1 none/minimal, 2 quitting groups, 2 continuous groups) and 7 smoking trajectories (1 none/minimal, 2 quitting groups, 4 continuous groups). Women with pregnancies assigned to the low- or high-continuous drinking groups were less likely to have completed high school and were more likely to have enrolled in the study in the third trimester, be of mixed ancestry, or be depressed than those assigned to the none/minimal or quit-drinking groups. Results were similar when comparing continuous smokers to none/minimal and quit-smoking groups. Further, women classified as high- or low-continuous drinkers were more likely to smoke at moderate-, high-, and very high-continuous levels, as compared to women classified as non-drinkers and quitters. This is the first study of this size to utilize group-based trajectory modeling to identify unique prenatal drinking and smoking trajectories. These trajectories will be used in future analyses to determine which specific exposure patterns subsequently manifest as poor peri- and postnatal outcomes.

Chemotaxis: new role for Ras revealed

A recent study of chemotaxis revealed a new role for the proto-oncogene Ras in the social ameba Dictyostelium discoideum. Chemotaxis, the directional movement of cells toward chemokines and other chemoattractants, plays critical roles in diverse physiological processes, such as mobilization of immune cells to fight invading microorganisms, targeting of metastatic cancer cells to specific tissues, and guidance of sperm cells to ova during fertilization. This work, published in the July 26 issue of The Journal of Cell Biology, was conducted in Dr. Devreotes’ lab at John Hopkins University and Dr. Parent’s lab at National Cancer Institute. This research team demonstrated that RasC functions as an upstream regulator of TORC2 and thereby governs the effects of TORC2-PKB signaling on the cytoskeleton and cell migration. Many of the core components of the underlying chemotaxis signaling network have been elucidated in D. discoideum. Chemoattractants are sensed by G-protein-coupled receptors (GPCRs), which leads to the activation heterotrimeric Gproteins, small Ras-like G-proteins, and phosphoinsositide 3kinase (PI3K), resulting in the generation of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). This phospholipid, in turn, prompts the membrane translocation of proteins containing pleckstrin homology (PH) domains, such as cytosolic regulator of adenylyl cyclase (CRAC) and protein kinase B (PKB), which regulate the cytoskeleton rearrangements during chemotaxis. Importantly, many of these components and their activation are highly localized to the leading edge of cells undergoing chemotaxis, assuring that cytoskeletal changes needed for directional movement are spatially restricted (reviewed in Jin et al., 2009). Although it has been well established that the PIP3 pathway plays an important role in the regulation of chemotaxis, additional pathways that act in parallel with the PIP3 pathway have recently been revealed. For instance, phospholipase A2 (PLA2) was reported to mediate chemotaxis in parallel with PIP3 pathway (Chen et al., 2007). In addition, a PIP3independent pathway in which PKB is activated by TORC2 (target of rapamycin complex 2) was found to regulate chemotaxis (Lee et al., 2005; Kamimura et al., 2008). However, the mechanism by which TORC2 is regulated in chemotaxis was poorly understood prior to the publication of the recent report by Cai et al. (2010). D. discoideum possesses two PKB homologs, namely PKBA, which contains a PH domain and is dynamically recruited to the plasma membrane by PIP3, and PKBR1, which is tethered to the plasma membrane via N-terminal myristoylation. In their previous work, the authors discovered that both PKBA and PKBR1 are activated by TORC2mediated phosphorylation of their hydrophobic motifs (HMs) (Kamimura et al., 2008) and phosphoinositide-dependent kinase (PDK)-mediated phosphorylation of their activation loops (ALs) (Kamimura and Devreotes, 2010). In the present study, Cai and colleagues investigated whether Ras family proteins activate TORC2 and, if so, what are the effects of this Ras-TORC2 pathway on chemotactic responsiveness. In order to examine whether Ras proteins are required for TORC2-mediated activation of PKB, the scientists first determined the PKB activity in different Ras knock-out cells. They found that phosphorylation of the HM of PKBR1, the ALs of both PKBR1 and PKBA, and many PKB substrates were significantly reduced in rasC but not rasG cells relative to wild-type cells. To further explore rasCs role in PKB activation, the authors examined the consequences of expressing activated RasC (RasC) and found that it dramatically prolonged the phosphorylation kinetics of the PKBR1 and multiple PKB substrates, suggesting that RasC is indeed involved in regulating the PKB pathway. In addition, RasC expression also prolonged actin polymerization and impaired chemotaxis in wild-type cells, effects that were suppressed in cells lacking Pianissimo (piaA), an essential component of TORC2. Taken together these findings

Epigenetics, Alcohol, and Cancer

Chronic or excessive alcohol consumption often leads to various medical disorders, including liver cirrhosis, pancreatitis, cardiomyopathy, fetal abnormalities, brain damage, and various cancers. To effectively prevent or treat these disorders, a better understanding of their underlying mechanisms is pivotal. Alcohol and its metabolites alter cellular functions through changes at the levels of DNA, RNA, protein, and metabolites, resulting in pathophysiological changes. Although the amount of RNA, protein, or metabolites is determined by dynamic processes of synthesis and degradation with many regulators involved, it is initially predominantly determined by the activity of the genes involved. Two major mechanisms of gene regulation involve genetic and epigenetic controls of gene expression. In the past half century, research and technological innovations have led to a remarkable progress in understanding the genetic controls of gene activities. However, only in recent years, epigenetic controls of gene expression drew immense interest, especially in the biomedical fields of cancer and ageing. To date, research efforts to understand effects of alcohol on regulation of gene activity have been primarily focused on genetic factors. The mechanisms of epigenetic modifications in alcohol-induced organ damage, including cancers, have only started to be tackled in the past few years.