Project description:The ability of a cell to regulate and adapt its internal state in response to unpredictable environmental changes is called homeostasis and this ability is crucial for the cell's survival and proper functioning. Understanding how cells can achieve homeostasis, despite the intrinsic noise or randomness in their dynamics, is fundamentally important for both systems and synthetic biology. In this context, a significant development is the proposed antithetic integral feedback (AIF) motif, which is found in natural systems, and is known to ensure robust perfect adaptation for the mean dynamics of a given molecular species involved in a complex stochastic biomolecular reaction network. From the standpoint of applications, one drawback of this motif is that it often leads to an increased cell-to-cell heterogeneity or variance when compared to a constitutive (i.e. open-loop) control strategy. Our goal in this paper is to show that this performance deterioration can be countered by combining the AIF motif and a negative feedback strategy. Using a tailored moment closure method, we derive approximate expressions for the stationary variance for the controlled network that demonstrate that increasing the strength of the negative feedback can indeed decrease the variance, sometimes even below its constitutive level. Numerical results verify the accuracy of these results and we illustrate them by considering three biomolecular networks with two types of negative feedback strategies. Our computational analysis indicates that there is a trade-off between the speed of the settling-time of the mean trajectories and the stationary variance of the controlled species; i.e. smaller variance is associated with larger settling-time.
Project description:According to medical guidelines, the distinction between "healthy" and "unhealthy" patients is commonly based on single, discrete values taken at an isolated point in time (e.g., blood pressure or core temperature). Perhaps a more robust and insightful diagnosis can be obtained by studying the functional interdependence of such indicators and the homeostasis that controls them. This requires quasi-continuous measurements and a procedure to map the data onto a parsimonious control model with a degree of universality. The current research illustrates this approach using glucose homeostasis as a target. Data were obtained from 41 healthy subjects wearing over-the-counter glucose monitors, and projected onto a simple proportional-integral (PI) controller, widely used in engineering applications. The indicators quantifying the control function are clustered for the great majority of subjects, while a few outliers exhibit less responsive homeostasis. Practical implications for healthcare and education are further discussed.
Project description:Cholesterol is required to maintain the functional integrity of cellular membrane systems and signalling pathways, but its supply must be closely and dynamically regulated because excess cholesterol is toxic. Sterol regulatory element-binding protein 2 (SREBP2) and the ER-resident protein HMG-CoA reductase (HMGCR) are key regulators of cholesterol biosynthesis. Here, we assessed the mechanistic aspects of their regulation in hepatic cells. Unexpectedly, we found that the transcriptionally active fragment of SREBP2 (N-SREBP2) was produced constitutively. Moreover, in the absence of an exogenous cholesterol supply, nuclear N-SREBP2 became resistant to proteasome-mediated degradation. This resistance was paired with increased occupancy at the HMGCR promoter and HMGCR expression. Inhibiting nuclear N-SREBP2 degradation did not increase HMGCR RNA levels; this increase required cholesterol depletion. Our findings, combined with previous physiological and biophysical investigations, suggest a new model of SREBP2-mediated regulation of cholesterol biosynthesis in the organ that handles large and rapid fluctuations in the dietary supply of this key lipid. Specifically, in the nucleus, cholesterol and the ubiquitin-proteasome system provide a short-loop system that modulates the rate of cholesterol biosynthesis via regulation of nuclear N-SREBP2 turnover and HMGCR expression. Our findings have important implications for maintaining cellular cholesterol homeostasis and lowering blood cholesterol via the SREBP2-HMGCR axis.
Project description:Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of ?-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation) are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for 'synthetic cellular heterogeneity' that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism), demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a 'phenotypic sensitivity analysis' method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in isolation, and its impact on overall system performance once integrated.
Project description:As we begin to design increasingly complex synthetic biomolecular systems, it is essential to develop rational design methodologies that yield predictable circuit performance. Here we apply mathematical tools from the theory of control and dynamical systems to yield practical insights into the architecture and function of a particular class of biological feedback circuit. Specifically, we show that it is possible to analytically characterize both the operating regime and performance tradeoffs of an antithetic integral feedback circuit architecture. Furthermore, we demonstrate how these principles can be applied to inform the design process of a particular synthetic feedback circuit.
Project description:Integral feedback control is a basic engineering strategy for ensuring that the output of a system robustly tracks its desired value independent of noise or variations in system parameters. In biological systems, it is common for the response to an extracellular stimulus to return to its prestimulus value even in the continued presence of the signal-a process termed adaptation or desensitization. Barkai, Alon, Surette, and Leibler have provided both theoretical and experimental evidence that the precision of adaptation in bacterial chemotaxis is robust to dramatic changes in the levels and kinetic rate constants of the constituent proteins in this signaling network [Alon, U., Surette, M. G., Barkai, N. & Leibler, S. (1998) Nature (London) 397, 168-171]. Here we propose that the robustness of perfect adaptation is the result of this system possessing the property of integral feedback control. Using techniques from control and dynamical systems theory, we demonstrate that integral control is structurally inherent in the Barkai-Leibler model and identify and characterize the key assumptions of the model. Most importantly, we argue that integral control in some form is necessary for a robust implementation of perfect adaptation. More generally, integral control may underlie the robustness of many homeostatic mechanisms.
Project description:PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.
Project description:SUMOylation-protein modification by the small ubiquitin-related modifier (SUMO)-affects several cellular processes by modulating the activity, stability, interactions or subcellular localization of a variety of substrates. SUMO modification is involved in most cellular processes required for the maintenance of metabolic homeostasis. Cholesterol is one of the main lipids required to preserve the correct cellular function, contributing to the composition of the plasma membrane and participating in transmembrane receptor signalling. Besides these functions, cholesterol is required for the synthesis of steroid hormones, bile acids, oxysterols and vitamin D. Cholesterol levels need to be tightly regulated: in excess, it is toxic to the cell, and the disruption of its homeostasis is associated with various disorders like atherosclerosis and cardiovascular diseases. This review focuses on the role of SUMO in the regulation of proteins involved in the metabolism of cholesterol.
Project description:Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.
Project description:Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.