Educational Outreach: Symposium 2003 Speakers

Dr. Joseph L. Goldstein
Dr. Lucy Shapiro
Dr. John Potter
Dr. Ken Stuart
Dr. Leonid Kruglyak
Dr. Alan Aderem
Dr. Rafi Ahmed
Dr. Raimond Winslow
Dr. Richard Young

Speaker:
Dr. Joseph L. Goldstein

Presentation Title:
Regulated Intramembrane Proteolysis (RIP): An Ancient Signaling Process, Newly Uncovered

Regulated intramembrane proteolysis (RIP) is a newly recognized mechanism for signal transduction that involves the proteolytic generation of regulatory molecules from membrane proteins. Such proteolytic cleavage liberates cytoplasmic or luminal/extracellular fragments of transmembrane precursor proteins, allowing the cleaved fragments to function at a new location. RIP is one of the most ancient mechanisms of signal transduction, operating in virtually all organisms (archaea, bacteria, plants, and animals); it controls cellular events as diverse as cholesterol metabolism and the unfolded protein response; and it plays a causative role in diseases from A to Z, atherosclerosis to Alzheimer’s. What makes RIP unique is that it is carried out by a class of membrane-bound proteases that cleave their protein substrates at forbidden sites -- forbidden in the sense that the sites of cleavage lie within membrane-spanning segments, which should be resistant to proteolysis because of the shielding effect of the hydrophobic environment of the lipid bilayer. At least 10 membrane proteins are currently known to undergo RIP, and their intramembrane cleavage is mediated by four different families of membrane-bound proteases. In this lecture, I will discuss how RIP controls cholesterol homeostasis through the proteolytic processing of a membrane-bound transcription factor called SREBP. I will also elaborate on RIP as a general biological process of cell signaling.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Lucy Shapiro

Presentation Title:
The Cell Cycle: Spatial and Temporal Control of a Multicomponent Genetic Network

Our goal is to define the complete genetic circuitry that coordinates cell differentiation as a function of a bacterial cell cycle. We study the bacterium, Caulobacter crescentus, which has a genome of 3767 gene with a clearly defined cell cycle. Microarray analysis revealed that the transcription of approximately 19% of the genome is cell cycle controlled. We found that in Caulobacter, as in yeast (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding protein that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA response regulator, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes and, of these, 55 operons (encompassing 95 genes) are directly controlled by CtrA. Among the CtrA regulated genes are 14 genes encoding regulatory proteins that are likely to regulate additional genetic modules yielding a serially connected loop of activators and repressors. Analysis of cell cycle regulatory factors revealed that temporally regulated phosphorylation cascades and proteolysis events are critical determinants of bacterial cell cycle control. In addition, we have recently shown that both chromosomal regions and regulatory proteins exhibit dynamic localization that is important for cell cycle progression. Thus, deciphering the entire regulatory network requires the coordination of multiple levels of control, including the integration of three-dimensional information.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. John Potter

Presentation Title:
Genetics and Exposures: Towards the Last Cohort Study

We now have a draft of the complete human genome. We will soon be able to characterize genotypes and haplotypes essentially to any degree of fine detail.

We are adapted to a wide variety of different environments, cultures, diets (both marginal and excessive), micro-organisms and parasites, toxic exposures, and bad habits, adapted at least sufficiently to survive to reproduce and raise the next generation. We are outbred and the variety of susceptibilities to, and protection against, these above exposures is marked. We suffer a significant number of diseases that impair quality and quantity of life and that are caused by exposure to these widely varying environmental exposures.

The accurate characterization and measurement of many of these exposures is difficult but there is an extensive body of skills and experience available. Much of the genetic variation that is associated with cancer risk is relevant only in the presence of environmental variability. Accordingly, what is needed involves not just measurement of genetic variation but also variation in environmental exposures.

The characterization of the disease phenotypes is still problematic, with a myriad classification schemes for different organs and systems ranging from the precise molecule to the vague syndrome.

To attempt to establish the complete pattern of human disease susceptibility and resistance, to identify more precise phenotypes, we need a large number of individuals
who are well characterized genetically, whose exposures are well mapped, and whose illness pattern and mortality can be monitored. What is proposed is a cohort study - a very large, ethnically diverse cohort on whom we have a substantial exposure history, family history, blood, and the capacity to follow up actively and passively for outcomes of interest.

The collected exposure data will be inexhaustible. Immortalized cell-lines or, if technology permits, established individual genome sequences, similarly, will be an inexhaustible biologic or electronic resource. Appropriate follow-up procedures will capture essentially all of the desired endpoints – and, with application of appropriate methods, their homogeneous molecular subtypes. Thus, we will have an unlimited resource for an extensive range of studies available to a large number of investigators.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Ken Stuart

Presentation Title:
Trypanosome RNA editing: Pathogen-specific RNA processing that controls energy generation

RNA editing is a form of post-transitional processing that inserts and deletes Us in mitochondrial pre-mRNAs in trypanosomatid pathogens to produce mature functional mRNAs. The edited sequence is specified by small guide RNAs (gRNAs) and occurs by a series of coordinated catalytic steps. The catalytic steps are performed by a multi-protein complex, the editosome. We have identified numerous editosome protein components and find that they occur as families or pairs of related proteins. A combination of biochemical and genetic approaches identified the functions of several proteins, and also identified specific interactions among these proteins. Surprisingly, it is emerging that the editosomes are physically and functionally organized into insertion and deletion sub complexes, although the functional separation appears incomplete. Despite the ability of rare mutant trypanosomatids to survive in some life cycle stages, regulated inactivation of editing is lethal in these stages. Hence editing is normally an essential function in these pathogens and thus is a possible target for drug development. However. these organisms appear to have the ability to compensate for the loss of this essential function.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Leonid Kruglyak

Presentation Title:
Mapping Natural Variation in Gene Expression

Several studies have shown that natural genetic variation can cause significant differences in gene expression, suggesting that phenotypic diversity results not only from coding variation that affects protein function but also from regulatory variation that affects gene expression. However, little is known regarding the mechanisms by which natural polymorphisms affect gene regulation. To begin to understand the genetic architecture of natural variation in gene expression, we carried out genetic linkage analysis of genome-wide expression patterns in a cross between a laboratory and a wild strain of Saccharomyces cerevisiae. Between a quarter and a half of all genes were differentially expressed between the parent strains. Expression levels of over 2200 genes were linked to one or more different loci, with most expression levels showing complex inheritance patterns. Most linkages were to one of two types of loci: cis-acting modulators of single genes and trans-acting modulators of many genes. We found 13 such trans-acting loci, each affecting expression of a large group of genes of related function. Using a recently published collection of transcription factor binding sites, we found that a number of expression clusters of genes were significantly enriched for targets of particular transcription factors, explaining their coordinate regulation. However, variation in expression of genes within a cluster mapped to the location of the corresponding transcription factor in only one case. More generally, for all genes showing highly significant linkage, very few linked to a transcription factor of which they were a target, and there was no tendency for all linkages to fall near a transcription factor rather than elsewhere in the genome. These results suggest that regulatory genetic variation typically occurs at nodes other than transcription factors in the regulatory network.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Alan Aderem

Presentation Title:
Macrophage activation in innate immune responses: from genes to function

The immune system of vertebrates consists of innate and adaptive components that differ in their mechanisms of pathogen recognition and the immediacy of their responses. The innate immune system is the first line of defense. It uses germ line-encoded pattern-recognition receptors that recognize the conserved molecular structures expressed in pathogens, but not in the host. The adaptive immune system has exquisite specificity mediated by antigen receptors that are generated by gene rearrangement. However, the development of an effective adaptive response takes time, and it is governed by elements of the innate immune system.

Macrophages, and the related dendritic cells, represent the cornerstone of the innate immune system. On recognizing a pathogen, the macrophage engulfs it and then undergoes a program of activation: this results in a differentiated cell that is capable of killing the microbe and initiating the adaptive immune response through antigen presentation and cytokine production. These activities must be tightly regulated since an inappropriate, or excessive, response leads to inflammatory disease. The control of the balance between host defense and inflammatory disease is very complex, and results from the integration of a large number of inputs: these inputs involve cross-talk by many receptors on the cell surface. These include pattern-recognition receptors, phagocytic receptors, and cytokine receptors, to name just a few. The mechanism, by which complex inputs are converted to appropriate inflammatory responses, can only be studied by systems approaches, and this will be the focus of my presentation.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Rafi Ahmed

Presentation Title:
Immunological Memory: Remembering Our Pathogens

Acute viral infections induce long-term humoral and cellular immunity. However, the nature of T- and B-cell memory is different. Antiviral B-cell memory is usually manifested by continuous antibody production that lasts for many years after infection or vaccination. In contrast, the effector phase of the T cell response is short-lived (a few weeks), and “memory” in the T-cell compartment results from the presence of memory T cells, which are found at higher frequencies and can respond faster and develop into effector cells (i.e., CTL or cytokine producers) more efficiently than can naïve T cells. In this talk I will discuss the following aspects of immunological memory: (1) The importance of long-lived plasma cells in maintaining humoral immunity. (2) Functional differences between naïve and memory T cells (3) Memory T cell differentiation and memory cell subsets; and (4) Protective immunity by memory CD8 T cells.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Raimond Winslow

Presentation Title:
Systems Biology: A Necessary Methodology for Understanding the Mechanisms of Sudden Cardiac Death in Heart Failure

Heart failure (HF), the most common cardiovascular disorder, is characterized by ventricular dilatation, decreased myocardial contractility and cardiac output. Prevalence in the general population is over 4.5 million, and increases with age to levels as high as 10%. New cases number approximately 400,000 per year. Patient prognosis is poor, with mortality roughly 15% at one year, increasing to 80% at six years subsequent to diagnosis. It is now the leading cause of Sudden Cardiac Death (SCD) in the U.S. Our studies of heart failure use a well known animal model of this disease – the canine tachycardia pacing-induced heart failure model. With the recent funding of the JHU Canine Microarray Facility and the JHU-NHLBI Proteomics Center, we now have the ability to undertake a comprehensive study of disease mechanisms by collecting comprehensive data sets that describe changes in: a) gene expression; b) protein expression level and post-translational modifications; c) cellular electrophysiology; d) cardiac micro-anatomic structure; and e) cardiac electrical conduction. It is our intent to use quantitative models as the framework by which to interpret data collected at each of these levels. This talk summarizes our progress to date.

We will begin by presenting an experimentally-based computational model of normal versus failing myocytes, and show how this model was used to predict that changes in expressed levels of genes/proteins involved in excitation-contraction (EC) coupling are a major contributor for risk of arrhythmia in heart failure. This model prediction has subsequently been confirmed through experimental studies and has important implications for disease treatment. We will also show how this pro-arrhythmic effect is mediated by highly localized interactions between individual L-Type Ca2+ channels in the sarcolemmal membrane and clusters of ryanodine-sensitive Ca2+-release channels in the junctional sarcoplasmic reticulum membrane. This is a fascinating example of how microscale interactions between stochastically gating channels has a profound impact on the macroscopic behavior of the cardiac myocyte.

Heart failure is accompanied by profound changes of ventricular geometry and fiber structure. We will describe how the micro-anatomic structure and geometry of normal and failing hearts may be measured using diffusion tensor magnetic resonance imaging (DTMRI). We will show two approaches to analyses of these structural data. First, we will describe a relational database system that enables users to pose queries regarding structural differences between hearts by means of visual interaction with a 3-D display. Second, we will present new techniques for performing formal hypothesis testing on differences in anatomic shape and structure, and then illustrate how these techniques may be applied to identify statistically significant changes in the geometry and fiber structure of normal versus failing hearts. We then show how structural data obtained from DTMRI studies and biophysical data from electrophysiological studies may be combined to formulate large-scale models of electrical conduction in normal versus failing hearts, and how these models may be used to demonstrate a possible mechanism by which changes in expression levels of EC coupling genes/proteins contribute to whole-heart arrhythmia. Finally, we will conclude by showing recent results indicating it is now possible to measure electrical conduction on the epicardial surface of a beating heart using high-density sock electrode arrays, to then image and model that heart, and to then compare model conduction patterns with those measured experimentally in the same heart. This is a critically important step towards closing the loop between experimental measurements and modeling data at the whole-heart level. (supported by NIH RO1-HL60133, RO1-HL70894, RO1-HL72488, NO1-HV-28180, P50 HL52307, the Whitaker Foundation, the Falk Medical Trust and IBM Corporation.)

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak
Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top

Speaker:
Dr. Richard Young

Presentation Title:
Transcriptional Regulatory Networks in Living Cells

Gene expression programs depend on recognition of specific promoter sequences by transcriptional regulatory proteins, which recruit and regulate chromatin modifying complexes and components of the transcription apparatus. We are determining the distribution of transcriptional regulators and chromatin modifying complexes throughout the genome in yeast and human cells. We use new algorithms that combine this information with gene expression data to develop and test models of the transcriptional regulatory networks that control cellular processes.

Dr. Joseph L. Goldstein Dr. Lucy Shapiro Dr. John Potter Dr. Ken Stuart Dr. Leonid Kruglyak

Dr. Alan Aderem Dr. Rafi Ahmed Dr. Raimond Winslow Dr. Richard Young Top