Several of the NICHD Fellows Award for Research Excellence (FARE) winners who will present at this year’s NIH Research Festival (October 24-28) have collaborated with The NICHD Connection to bring you a blurb about their research—understandable to everyone, whether you are a biologist, physicist, or clinician! If something piques your curiosity, check out their talk or poster at the festival!
In no particular order, the award-winning research of this year’s NICHD FARE recipients:
Criminal Activities of Bacteria Revealed—Legionella Grand Theft Auto
By Yang Chen, Graduate Exchange Student from Peking University, Beijing
Legionnaire’s disease, a severe form of pneumonia, sends nearly 8,000-18,000 people to the hospital each year, according to the Centers for Disease Control. The bacteria Legionella pneumophila causes Legionnaires' disease by infecting alveolar macrophage, an important cell type that resides in the surface layer of the lung alveoli. The bacteria hijack intracellular vesicle transport (the mechanism used by the cell to shuttle proteins around) and establish a camouflaged compartment resembling host-cell rough endoplasmic reticulum (the cellular machinery used to make and process proteins) to conceal the bacteria’s own replication.
Legionella perpetrates its criminal activities in several ways, one of which involves transiently exploiting the activity of a key regulator of protein transport, called Rab1. The bacteria produce another protein, called SidM, which takes the Rab1 protein hostage and locks it in an active conformation through a process called AMPylation. With Rab1 as its hostage, the bacteria can elicit control over protein transport in the cell.
Most recently, we have identified how the bacteria convert Rab1 back into a non-AMPylated state---a prerequisite for the efficient use of Rab1’s talents. Legionella use SidD, the first known de-AMPylase among bacterial virulent factors.
By mutating single pieces—one at a time—of the SidD protein, we identified an activity domain at the beginning of SidD and a targeting domain at the end of the protein. These findings offer insight into the molecular mechanism of SidD-based activities. Without SidD, the Rab1 hostage cannot be fully utilized in Legionella infected cells, indicating the importance of SidD for Legionella’s transportation theft!
Does Legionella use reversible modifications of hostage proteins to manipulate protein transport activities for Legionella’s own benefits? Our verdict: guilty as charged!
Don’t Judge a Protein by Its Size—The Story of YneM
By Xuefeng Yin
In the past, researchers had ignored proteins of less than 50 amino acids within organisms. They were excluded in initial genome annotation and missed in classical genetic and biochemical characterization. Recent studies, however, have revealed an increasing number of small proteins that are critical to the bacterial cell. Small proteins have diverse roles, acting as: signal transducers, metal and nucleic acid chaperones, stabilizing factors for larger complexes, and adaptors for protein degradation. The conserved 31-amino acid protein called YneM is a good example that even the little guy can play a critical role in the cell.
YneM, identified in the bacteria E. coli, sits in the cytoplasmic membrane where it connects magnesium and phosphate regulation and possibly modulates their transport. YneM is highly produced when the magnesium level is low. It associates with a phosphate transporter in the membrane and potentially promotes export of phosphate through this transporter, thereby causing a drop in phosphate level inside the cell. This may release magnesium from the phosphate bound form and make magnesium ions available for enzymes to use.
The role of YneM exemplifies the idea that small proteins can accumulate under specific conditions and quickly respond to harmful environments at a relatively low energy cost. Generating larger proteins in response to a stressful condition would use a lot of energy for the cell, so in this case, maybe smaller really is better!
The Mystery of Brain and Liver Dysfunction in Niemann-Pick disease type C
By Celine Cluzeau, PhD
Niemann-Pick disease type C (NPC) is an inherited disorder characterized by cholesterol and fat build-up in the cells of the liver, spleen, and brain. This build-up leads to liver disease, progressive neurodegeneration, and ultimately, lethality. The root problem of the disease resides with a mutation in one of the NPC1 or NPC2 genes, which renders the cell compartments responsible for breaking down cholesterol and fats unable to complete the task. To date, there is no FDA approved therapy for NPC. Despite intensive study, the mechanisms leading to both brain and liver dysfunctions are poorly understood.
All cells function according to the genes that are being used. To determine why cells in NPC do not function properly, we compared which genes were being turned up or down in a mutant mouse model that lacked all NPC1 production (and closely recapitulated the human disorder) to the gene expression of healthy mice.
Upon studying the gene expression levels every other week, spanning the full disease progression from the first week to the terminal disease time of 11 weeks, we identified 222 altered pathways, including metabolic processes, immune responses and developmental signaling pathways. Among the genes that were differentially expressed at all ages, nine genes in the cytochrome P450 (CYP) family—enzymes that are involved in the metabolism of drugs and other intracellular compounds—were turned down in the mutant mice. CYP enzyme downregulation is a significant pharmacogenetic finding, since impaired CYP activity may result in altered drug metabolism by NPC patients and thus require alternative medication dosing.
Our findings are supported by preliminary data in mouse model, feline model, and NPC patients’ livers, where there is a reduction of CYP activity in vitro. We are currently investigating CYP activity in vivo in the mouse model. In parallel, we are also working on the altered genes and pathways we identified with two objectives: 1) identifying proteins that we could use as biomarkers for NPC, and 2) understanding the link between these altered pathways and NPC presentation, to discover potential drug targets.
Early Warning System to Predict If Cancer Will Spread
By Saravana Murthy, PhD
Cancer is a leading cause of death worldwide. The World Health Organization estimates that cancer will contribute to over 11 million deaths by 2030. In an intriguing twist, the initial tumor is likely not the deadly culprit. Rather, fatal scenarios arise after the primary tumor spreads to vital organs in a process called metastasis.
Clinicians are vigorously searching for a metastasis warning system. Traditional diagnostics rely on morphological and histological analysis, which are not very accurate and fail to identify and/or distinguish high risk and low risk patients for metastasis. These misinterpretations could lead to treatment failure and cause early fatality. Genetic biomarkers, however, could significantly improve these predictions.
The biomarker CPE-delta-N may be an important tool in a clinician’s diagnostic toolbox. CPE-delta-N predicts the likelihood of tumor metastasis with accuracy rates approaching 90%. Tumor samples from 180 men and women with liver cancer showed that a doubling in the amount of CPE-delta N in the tumor, as compared to the surrounding tissue, predicted that the cancer was more likely to return or spread within two years. Similar results were found with pheochromocytoma (tumor of the adrenal glands), paraganglioma (neuroendocrine tumor), colorectal carcinoma and thyroid cancer. In fact, CPE-delta-N is not only a predictive marker for metastasis, it’s also a root of the problem. In mouse models, CPE-delta-N drives another key gene called NEDD9 to induce tumor metastasis.
Validation of the CPE-delta-N biomarker in a larger cohort of patients will hopefully lead to a future in which a patient’s CPE-delta N levels could be used to guide individualized cancer care.
Mathematical Modeling of Membrane Pit Formation
By Anand Banerjee, PhD
Cells use a special process, called endocytosis, to engulf external material and package it into a transportable vesicle. To protect the vesicle, the cell uses a protein, called clathrin, which forms a highly-structured protective protein lattice. Clathrin mediated endocytosis (CME) is of fundamental importance to organisms in many ways, including—but not limited to—providing nutrition, regulating cholesterol metabolism and responding to hormone signals.
CME begins with the assembly of specialized proteins, including clathrin, on the internal side of the cell membrane. The protein assembly aids in the formation of small membrane invaginations called clathrin-coated pits (CCPs). Experiments show that there is considerable variability in the dynamics of CCPs. Some CCPs exist for only a few seconds and then disassemble abruptly. Other CCPs are relatively long-lived and grow steadily in size to form a vesicle. Even among the long-lived ones, there is a large variance in size and lifetimes. The origin of this heterogeneity is not clearly understood.
I am currently working on the development of a mathematical model to describe CCP assembly. My aim is to estimate the extent to which the stochastic nature of protein assembly is responsible for the observed variation in CCP dynamics.
Examining an Interneuron’s Journey
By Brian Erkkila, PhD
Information processing by the brain and the rest of the central nervous system (CNS) is finely tuned by the interplay of excitatory and inhibitory neurons. Within the CNS, the hippocampus is a structure responsible for learning and memory, and it is no surprise that this region is of considerable importance in the pathologies of epilepsy, schizophrenia and Alzheimer’s disease. The inhibitory neurons, or interneurons, of this region are not generated in their final locations in the neural circuit, but rather in a distinct brain region known as the ganglion eminence. During embryonic development, these interneurons migrate by “crawling” through the cortex until they ultimately reach their final destination in the hippocampus. Details about interneuron relocation have yet to be described.
We used genetically engineered mice that have a population of these interneurons labeled with a green fluorescent protein to track interneuron migration. Early in development, on embryonic day 12 (E12), the journey to the hippocampus takes up to four days. Closer to birth at E16, however, the journey takes only two days—despite the fact that the embryonic animal’s brain has grown considerably and that the interneuron has had further to travel. In addition, we found that there was a significant reduction in the number of interneurons after birth. In fact, between birth and postnatal day 10, there was an ~80% reduction in the number of interneurons found in the hippocampus. Our future studies will focus on the attractive and repulsive cues responsible for interneuron migration as well as the identities of the interneurons lost during the first days of life.
How to Make Frog Gut Stem Cells from Tadpoles—Really!
By Kenta Fujimoto, PhD
One hundred years ago, Dr. J. F. Gudernatsch made the remarkable discovery that a substance in the thyroid gland could cause tadpoles to turn into frogs. Since Gudernatsch and others established that thyroid hormone (T3) is a developmental signal that triggers the onset of metamorphosis, scientists have used amphibians for studying the mechanism of T3 action and environmental toxicology.
T3 plays a major role in remodeling and organogenesis during postembryonic development, a period around birth in mammals when T3 levels are high. Amphibian metamorphosis resembles mammalian postembryonic development, giving us a unique opportunity to study T3 function during development.
During amphibian metamorphosis, the tadpole intestine (predominantly a monolayer of larval epithelium, or a densely packed continuous sheet of cells) exhibits a dramatic transformation. The larval epithelial cells undergo cell death, and concurrently adult epithelial stem/progenitor cells (unique cells that can divide and replace damaged intestinal tissue) develop de novo through an unknown mechanism. How epithelial cells in tadpoles are slated to become adult stem cells is an active area of investigation.
We have shown that the expression of a gene called T3 receptor-coactivator protein arginine methyltransferase 1 (PRMT1) is increased in the small number of tadpole epithelial cells that appear to give rise to the adult stem cells. We have analyzed how PRMT1 is specifically upregulated in the cells fated to become stem cells. Our findings suggest the involvement of the c-MYC transcription factor, a protein responsible for guiding which genes in the DNA are to be used. Interestingly, c-Myc is turned up during metamorphosis prior to the increase of PRMT1 in the intestine.
From tadpole to frog, we have used amphibian metamorphosis to show that stem cell specific expression of PRMT1, potentially affected by the activities of c-Myc, may be an important mechanism during adult intestinal stem cell development.
Manganese Meets Its “Match”
By Lauren Waters, PhD
Cells are complex and crowded systems, full of proteins, lipids, DNA, RNA, and many other molecules. Mediating the correct binding of any two molecules in such a crowded environment is a daunting task for a cell. The problem is even more challenging when discriminating between two similar substrates, such as between different metal ions. To ensure that the correct metal is inserted into important enzymes, cells are thought to employ dedicated protein carriers called metallochaperones. These proteins “match” metal-using enzymes with the right metal, thereby guaranteeing that the enzymes are active and fully functional. However, only a few such metallochaperones are known to date, and only for a handful of metals.
We have recently identified a new candidate metallochaperone for the poorly studied but essential metal manganese in the model bacterium E. coli. Manganese promotes survival during oxidative stress and is required for bacterial virulence during pathogenesis. We discovered a previously unknown small protein of only 42 amino acids that is regulated by manganese. Elevated levels of this small protein, called MntS, caused a growth defect in the presence of manganese, but not any other metals. We also found that MntS could bind to manganese and other proteins, leading us to hypothesize that MntS may be the elusive manganese chaperone.
In vivo studies are underway to test the function of known manganese-using enzymes, such as manganese superoxide dismutase (Mn-SOD), in the presence or absence of MntS. In addition, we have purified MntS and are performing in vitro biochemical studies to elucidate mechanism of action of MntS. Did manganese finally meet its match in the lab? Our work to answer this question will advance our knowledge of manganese homeostasis and the intracellular trafficking of this important metal.
The Building Blocks of Neuron Communication—It’s Not Babies’ Play
By Madhav Sukumaran, PhD
Neurons communicate at junctions, called synapses, where chemical messages bind to specialized receptor proteins on the receiving neuron’s membrane, thereby initiating an electrical response. One receptor subtype (the AMPA-type glutamate receptor) is responsible for fast, point-to-point communication between neurons. AMPA receptors are comprised of a number of subunits that can join together into different combinations, like Lego building blocks. These arrangements can influence the electrical properties of the neural connections and affect the function of the neuronal networks in which these neurons are embedded. The wrong configuration of AMPA receptor building blocks can contribute to the pathophysiology of such disorders as epilepsy, Alzheimer’s disease, and stroke. A complete understanding of the molecular mechanisms underlying AMPA receptor assembly is a major goal of this work.
Using high-resolution biophysical, electrophysiological, and crystallographic assays, we investigated the role of the AMPA receptor N-terminal domain (NTD), a section of the protein that was previously implicated in receptor assembly. We showed that the NTD plays an organizing role in the initial assembly steps and identified which NTD components controlled the assembly process, much like finding hidden marks on Legos that would indicate two pieces should fit together.
Not all AMPA receptor assemblies are created equal, so we explored the physiological and pathological conditions that lead to the formation of favorable and unfavorable receptor populations. Our investigations of the NTD molecular architecture revealed that the N-terminal domain may be more dynamic than previously thought and may have an additional role in the neuron: directly regulating receptor electrical function through potentially novel mechanisms. As the title suggests, these blocks are not for babies.
Cell Biology Is Like a Rock Concert
By Silviya P. Zustiak, PhD
Imagine a crowded rock concert. You are directly in front of the stage, but your cute date is stuck behind an enormous mass of moving people. As your date pushes and squeezes through the crowd, you worry about other attractive fans stealing your date’s attention. Will your date make it through all those packed people? Believe it or not, this is an important question in cellular biology!
The typical cell is crowded with both charged and neutral molecules that slow down or completely prevent passive movement, called diffusion, of soluble proteins through the cell (like your date moving through people at a rock concert). This crowding effect is implicated in all cellular processes. For example, crowding has been shown to trigger aggregation of a special class of proteins that are associated with neurotoxicity in the neurodegenerative disorder Alzheimer’s disease.
Despite vast interest in the subject, such hindered protein diffusion in the cell is still poorly understood. In other words, did your date fail to make it to the stage because there were too many people, or because your date found a more attractive person along the way?
In this project, we developed an in vitro cell model that has tunable binding and crowding to elucidate their relative roles on hindered protein diffusion. To simulate protein movement through a crowded cell, we labeled a small charged protein, called Ribonuclease A (RNase), with a fluorescent tag and monitored RNase diffusion through solutions of various charges (to simulate binding events) and various concentrations of dextran, a sugar polymer (to simulate crowding events).
In agreement with existing data, we observed a 5-fold decrease in RNase diffusivity in the most concentrated dextran solution. In this scenario, we measured that binding accounted for more diffusion inhibition than crowding. Further analysis revealed that one hundred times more crowder than binder was needed to achieve equivalent reduction in RNase diffusion. However, the data also suggested that at even higher crowder concentrations, similar to the levels found in a cell, crowding would overpower the effect of binding.
So what’s the bottom line? According to our analogy, if it’s a very densely packed concert, your date might not reach you due to the sheer number of individuals in the room. Otherwise, your date is likely to just get distracted by other cute people!