Overview of the PTN biology in Balch laboratory long-term goals
The key goals of the Balch laboratory are to (1) define how membrane trafficking pathways work and are tighly integrated with the proteostasis program to generate protein function, (2) determine the biochemical/molecular and structural roots of folding disorders that impact human healthspan such as cystic fibrosis and, (3) learn how therapeutic management of proteostasis biology can be used to restore the ability of the misfolded proteins to normal function to benefit human healthspan.
Through a multidisciplinary approach and application of diverse state-of-the-art cell biological, biochemical, molecular systems and structural approaches, we hope to gain critical insight into the fundamental principles of the integrated proteostasis cellular programs that manage both protein folding and trafficking, and a new understanding of the role of the proteostasis in controlling human health and aging.
We anticipate that knowledge of these pathways will enable the development of small-molecule PRs that adjust the folding environment to restore function and benefit human healthspan. Below we described the biological probein folding problem in the context of proteostasis and the multi-dimensional approaches we use to address the central role of proteostasis in human health and disease.
Human healthspan is now recognized to be critically sensitive to protein folding management. A major challenge is to understand how folding management keeps us healthy and how it responds to disease. Protein folding is managed by the 'protein homeostasis' or 'proteostasis' program (Balch et al. (2008) Science 319:916; Evans and Balch (2011) Nature, 471, 42). Proteostasis is essential to maintain the stem cell environment, to drive development, to protect us from the environment and to pathological challenges that occur daily, and during aging.
Inherited disease provides a unique challenge to the proteostasis system. All protein folding diseases arise as a consequence of an imbalance between the need for protein function, the energetics and kinetics of the protein fold and the properties of the local proteostasis managed folding environment that has both genetic and epigenetic foundations. Because up to 50% of all proteins encoded by the genome are likely at some point in their life cycle to reside the outside the cell, protein folding management is intrinsically linked to membrane trafficking pathways (Evans et al. (2009) Ann. Rev. Biochem. 78:959; Hutt and Balch (2010) Science, 329:766). These trafficking pathways include the extensive endomembrane system found in all eukaryote- both the exocytic and endocytic compartments. Thus, the proteostasis network (PN) involves numerous chaperone systems that are specialized for the cytosol and for each of the compartments of the endomembrane systems mediating trafficking generating what we refer to as the proteostasis trafficking network (PTN) which forms the 'cloud' around each protein to manage its daily function (Figure 1). Moreover, they are specialized for each cell type, and tissue and organismal environment (Evans and Balch (2012) Nature, In press). The PN includes the ubiquitous Hsp70 and Hsp90 chaperone/co-chaperone systems that both direct folding and protect the fold from genetic and physiological/pathological stresses that assault human physiology. Loss of proteostasis leads to major environmental triggered diseases including, among others, type 2 diabetes, COPD/emphysema, multiple amyloidoses including Alzheimer's-Parkinson's-Huntington's pathologies, and systemic (light chain) myeloma disease. Inherited misfolding disorders reflecting a mutation in the genetic code and an alteration in the polypeptide chain that compromise folding now contribute up to ~8,000 rare diseases world-wide reflecting the instability of the genome and the forces of Darwinian evolution (natural selection and fitness) and newly emerging Lamarckian-like epigenetic forces that tune the folding capacity through proteostasis daily to respond to the environment (e.g., survival) using histone acetylation and methylation pathways. Many inherited disorders including cystic fibrosis (CF) (see below), a prominent childhood disease, reflecting acquisition of missense, nonsense and/or deletion variants are broadly classified as 'membrane trafficking' disease because of a defect in protein folding during transit through the mammalian exocytic and/or endocytic trafficking pathways leading to a loss of normal function and/or a gain-of-toxic function that has major clinical impact.
Basis for the integrated misfolding and membrane traffic proteostasis system defining human biology
Eukaryotic cells are highly compartmentalized. Each compartment of the exocytic and endocytic pathways provides a unique chemical and biological environment in which protein folding and function can be modulated to maintain cellular, tissue and organismal homeostasis. During export from the first compartment of the exocytic pathway, the endoplasmic reticulum (ER) where folding is initiated, nearly one-third of the protein cargo encoded by the human genome is mobilized to the rest of the cell by the activity of vesicle budding machines that utilize tethering/fusion and coat components to direct membrane traffic. We now posit that these components are integral to the operation of the proteostasis machinery. In collaboration with Clint Potter and Bridget Carragher (Cell Biology), we have used cryo-electron microscopy (cryoEM) to solve the structure of the COPII coat- a self-assembling scaffold that contains an outer cage polymeric lattice responsible for collecting newly synthesized and folded protein cargo into the emerging budding vesicle- thus provided a link to the outside world. Not surprisingly, it is becoming increasingly apparent that the COPII coat assembly responsible for cargo selection and vesicle assembly and fission, and SNARE assemblies promoting vesicle tethering and fusion are intimately linked to components of the proteostasis program. For example, we and others have found that the selection of cargo clients such as CFTR (see below) (Figure 2) (Coppinger et al PLoS One (In press))and the components involved in the assembly and disassembly of coat and tethering systems is likely biologically regulated by the activity of the Hsp90 family of chaperone/co-chaperone components. Thus, the extensive machineries modulating rearrangements of the endomembrane system to accommodate and adjust the protein fold transiting endomembrane compartments are likely integral components of an under appreciated proteostasis-based trafficking network (PTN) (Figure 1) that manages the fold for function in diverse environments and in response to many challenges to human biology. By using bioinformatics and systems biology tools to understand network design, and by implementation of quantitative approaches to pathway dissection using mass spectrometry and modern sequencing technologies, we are beginning to define how the PTN begins to integrate the folding with trafficking to achieve the overall structure and function of the basic components facilitating endomembrane function.
Epigenetics and proteostasis in misfolding disease
Many mutations disrupt cargo traffic from the ER by preventing proper protein folding during synthesis in the ER resulting in loss of recognition by the COPII machinery. In collaboration with Jeffery Kelly (Chemistry) and Evan Powers (Chemistry) we have in the past used modeling approaches to develop a rigorous quantitative framework to describe in a global way the adaptable role of the TPN in health and disease. In collaboration with John Yates (Chemical Physiology), we are using novel mass spectrometry techniques to analyze the activity of the Hsp70 and Hsp90 chaperone/co-chaperone system that regulates the trafficking and function of a number of misfolding prone proteins responsible for inherited disease with a strong focus on the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that when defective is responsible for CF. Working with Joel Gottesfeld (Molecular Biology) and Reza Ghadiri (Chemistry), we have discovered that manipulation of the chromatin environment (the epigenome) through modulation of histone deacetylases (HDACs) using HDAC inhibitors (HDACi) or siRNA silencing of HDAC enzymes modulates a global network of interacting factors that contribute to restoration of the function of CFTR function in disease. These results suggest that transcriptional regulatory circuits controlled by gene silencing/unsilencing reactions such as histone acetylation/methylation pathways or post-translational modifications of non-histone proteostasis regulators such as Hsp90 may allow us to reprogram the diseased to a healthy state akin the operation of operation of evolutionary pathways that buffer change for fitness and survival. Moreover, our observations are consistent with the known effects of IgF-1-R growth factor receptor, caloric restriction and epigenetic modifiers that also influence the onset of other conformational diseases including type 2 diabetes and neurodegenerative amyloid diseases of aging that we are studying with our colleagues Jeffery Kelly, Joel Buxbaum (Experimental Medicine) and Andrew Dillin (The Salk Institute).
Pharmacological management of misfolding disease along the endomembrane axes- a new paradigm for disease correction
Taking advantage of our understanding of the molecular and biochemical relationships between proteostasis and PTN components and the biological functions they control, we are generating high-throughput-screening (HTS) approaches to monitor PN component contribution to folding and trafficking biology, and to identify small molecule regulators that precisely regulate key steps of critical PN pathways involved in human disease. One such area in the laboratory that has been showing significant progress is our recent discovery of small molecules that interfere with the function of the Hsp90 chaperone system and its extensive collection of co-chaperone regulators. These small molecules appear to have an important impact on the onset of misfolding disease. Thus, HTS approaches that target the PN are beginning to provide us with novel chemical and biological tools to understand and manage proteostasis biology.
Building a bioinformatic map of the PTN
In order to understand the many interactions governing the function of PTN in folding and trafficking management we are applying advanced bioinformatic tools to extensive databases acquired through various high-throughput-like experimental approaches to generate pathways that define healthy and unhealthy protein interactions, and how small molecules can adjust those pathways to achieve correction of function (Figure 3). The field of systems biology is in its infancy, and much work is necessary to create pictures of how a single protein operates in the context of healthspan to maximize organismal function, particular in response to disease.