Philip A. Rea

Our primary research activities center on the molecular biology, cellular biochemistry and proteomics of vacuolar function with special emphasis on membrane transport proteins and the enzymatic machinery responsible for the detoxification of xenobiotics, especially heavy metals.   Long-term objectives are to identify the proteins concerned and elucidate their mechanisms of action and regulatory characteristics.  The approach taken is that of the 'basic biologist' – the search for general principles, regardless of the organism in which they are to be elucidated, not just principles applicable to plants.  Many of the investigations conducted have therefore entailed parallel molecular and biochemical manipulations of several model systems including the plant Arabidopsis thaliana, the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans. It is by this approach that we have been able to make fundamental contributions toward understanding a remarkably broad range of transport and related phenomena of general significance.

A. Crystal structures of native ('Native NsPCS') and γ-Glu-Cys Cys70 thioester of Nostoc spp. phytochelatin synthase, NsPCS (‘γ-Glu-Cys-NsPCS’) (Vivares et al 2005).  In both cases one of the two monomers is shown as a ribbon structure; the other as the solvent accessible structure.  In the latter representation, red and gray patches correspond respectively to residues that are identical and similar between NsPCS and its eukaryotic homologs from A. thaliana, T. aestivum, Nicotiana tabacum, S. pombe and C. elegans.  Residues unique to NsPCS are shown in yellow.  Position of γ-Glu-Cys group in γ-Glu-Cys-NsPCS intermediate delimited by white circle.  B. Stereo view of NsPCS (structures and text in green) and papain (structures and text in gray) active site residues inferred from crystal structures (Vivares et al 2005).  Also indicated are the identities of the equivalent residues of AtPCS1 (in parenthesis as red text).  [Structures courtesy of Pascal Arnoux and David Pignol and adapted from Vivares et al (2005); active site residues first inferred from kinetic, site-directed and protein chemical investigations made by Rea and colleagues, reviewed in Rea (2012).]

a. ABC transporters

The ATP-binding cassette (ABC) protein superfamily is one the largest protein families known, and most, but not all, are membrane proteins (‘ABC transporters’) active in the transport of a broad range of substances across membranes.  Many of these proteins have been implicated in human diseases such as cystic fibrosis and Tangier disease, and resistance to therapies for cancer, malaria, and AIDS.  To quote from Higgins (2001):

The study of ABC transporters, in all their guises, has now become a minor industry. This is a far cry from ‘orphan’ beginnings, and provides a wonderful example of scientific serendipity, how fundamental studies of obscure model microbial processes, pretty much for their intrinsic interest with no obvious commercial or medical implications, can unexpectedly have a significant impact in unimagined arenas of biology. 

We have played a central role in the identification and molecular characterization of glutathione S-conjugate pumps (GS-X pumps) in yeast and plants. GS-X pumps, which belong to the multidrug resistance-associated protein (MRP) subfamily of ABC transporters , are involved in the vacuolar sequestration and/or plasma membrane extrusion and detoxification of both endogenous and exogenous toxins. Examples of substances transported by these membrane proteins are herbicides and heavy metals, as exemplified by cadmium and arsenic after their complexation with glutathione (GSH). As such, these transporters and their genes are of potential value for engineering plants with an increased capacity for the removal of toxic materials from contaminated soils and ground waters (Rea et al – US Patent No. 6,166,299). Current research is concerned with the analysis of Arabidopsis T-DNA insertion mutants for some of these transporters and in extending our understanding of the general significance of ABC transporters for plants by examining members of this superfamily implicated in the transport of folate (vitamin B9) and its derivatives, and other members implicated in the transport of iron-sulfur clusters across membranes for assembly of the prosthetic groups of oxidoreductases .  The overall impact of this work is illustrated by the fact that Arabidopsis allocates a large fraction of its open reading frames (a minimum of 0.5%) to members of the ABC superfamily.  We have assembled an inventory of more than 130 ORFs for ABC proteins in Arabidopsis, of which more than 100 are transmembrane proteins . This gene count far outstrips those for the human genome and any other animal genome sequenced.

b. phytochelatin-dependent heavy metal detoxification  

It has been known for some time that plants and some fungi synthesize peptides termed phytochelatins (PCs) from glutathione (GSH)  when exposed to heavy metals, and that PC thiols coordinate and chelate heavy metals to promote their removal from the cytosol by vacuolar sequestration.  However, the molecular identity of the enzyme(s) responsible eluded definition until the first cloning of the enzyme PC synthase (AtPCS1) from Arabidopsis by ourselves and two other groups. The isolation of AtPCS1 and its demonstrated sufficiency for PC synthesis from GSH both in vitro and in vivo has enabled detailed mechanistic analyses of this enzyme, and the provision of probes and methodologies for the identification and characterization of its orthologs in animals, as exemplified by our studies of C. elegans.

Although studies of PC synthases have largely been concerned with the enzymes from eukaryotes, recent database searches have disclosed PC synthase-like sequences in the genomes of several prokaryotes. In of itself, this finding might not be of particular interest except that all of the prokaryotic PC synthase homologs identified are half the length of their cognates from eukaryotes (220–237 residues compared with 421–506 residues) because they lack the more sequence-variable C-terminal domain . The one prokaryotic PC synthase homolog to have been assayed for activity, the alr0975 protein from the cyanobacterium Nostoc sp. PCC 7120 (NsPCS), catalyzes the deglycylation of GSH to γ-Glu-Cys at a high rate and the synthesis of PC2 at a relatively low rate. A recent crystal structure of NsPCS in its native and γ-Glu-Cys-acylated state (Vivares et al 2006) establishes, as had been inferred from our detailed kinetic, protein chemical, and site mutagenic analyses of the prototypical eukaryotic PC synthase, AtPCS1, that these enzymes belong to the papain superfamily and deploy a cysteine protease-like catalytic mechanism.

While this crystal structure, the first for a PC synthase, was indeed seminal, it will be imperative to acquire structural information for eukaryotic PC synthases either as C-terminal truncates or full-length molecules. Toward this end, it has been established that the N-terminal domain of AtPCS1, which undergoes Cd2+-independent γ-Glu-Cys acylation, is sufficient for the Cd2+-dependent synthesis of PCs from GSH whereas the C-terminal domain is necessary for two processes that are not essential for core catalysis, augmentative free Cd2+ sensing and γ-Glu-Cys acylation of the full-length enzyme at a second site .

When account is taken of the fact that in many plant species, PCs are the major ligands responsible for heavy metal accumulation and detoxification, AtPCS1 and genes like it may come to assume prominence in the development of phytoremediation technologies through genetic manipulation of the capacity of plants for heavy metal hyperaccumulation (Rea et al – US Patent No. 6,489,537). By the same token, the operation of equivalent metal detoxification pathways in nematodes and their cousins, some of which are pathogenic, may result in the development of new strategies for combating the many human and veterinary diseases caused by these organisms.

c. pyrophosphate-energized proton pumps

Our studies have been instrumental in elucidating the basic organization and core catalytic capabilities of proton-translocating inorganic pyrophosphatases (V-PPases), a novel class of proton pump. Membrane-associated proton-translocating PPases are primary proton pumps that use inorganic pyrophosphate (PPi), the limiting case of a high energy phosphate, instead of ATP as an energy source for the establishment of transmembrane electrochemical potentials . Although the initial objective of these studies was to understand the mechanism of this pump in plants, recent investigations in ours and other laboratories have demonstrated these pumps in organisms as disparate as thermophilic Archaea and parasitic protists. Among the many evolutionary, practical and bioenergetic implications of these findings is the possibility that this research will spawn new approaches to the treatment of several prolific and debilitating parasite-mediated infections.

It was originally thought that all V-PPases are of only one type, the type to which the first V-PPase to be cloned belongs. It is now clear that is not the case: V-PPases fall into two clearly delineated clades known as the type I and type II enzymes.  Type I and type II V-PPases are readily distinguished from each other on the basis of their sequences and the near obligate requirement of the former but not the latter for K+ for activity.  In Arabidopsis three genes, AVP1, AVP2, and AVP3 encode V-PPases.  AVP1 is the prototypical K+-dependent type I V-PPase which is expressed ubiquitously and at particularly high levels in developing tissues.  AVP2 and AVP3 are the type II V-PPases which share only 36% sequence identity with AVP1 but greater than 85% sequence identity with each other.

Given our success in isolating multiple knockout mutants for all three V-PPase genes in Arabidopsis, we are concerned with determining the physiological impact of this class of proton pump at the whole plant level.  At the time of writing, we have completed the first series of investigations of Arabidopsis knockout mutants of the gene encoding the canonical type I V-PPase, AVP1, and in so doing established its role in vacuolar anion accumulation and cytosolic pH stasis.

Now that we have a more accurate understanding of the role played by type I V-PPases in the intact plant – a platform on which the roles played by other V-PPases can be assessed on a comparative basis – we aim to better understand the roles played by the type II subclass in plants.  Is/are AVP2 and/or AVP3, unlike AVP1, freely reversible and able to catalyze H+-coupled PPi synthesis?  Does impaired type I and/or type II V-PPase function not only affect vacuolar compartmentation and general ionic stasis but also resistance to metabolic adversity?  Are type I and/or type II V-PPases involved in cellular high energy phosphate stasis and, if so, does this have repercussions for carbon metabolism?  These are the questions we are at long last in a position to address directly in a systematic and comprehensive manner.

d. vacuolar proteomics  

The vacuole of S. cerevisiae, which can occupy as much as 25% of total intracellular volume, participates in numerous cellular processes ranging from macromolecule degradation and salvage, pH and general ion homeostasis, osmoregulation and volume regulation, the storage of amino acids, carboxylic acids, carbohydrates and some vitamins, to the sequestration of endogenous and exogenous toxins. What is perhaps surprising given this multifunctionality is how little is known of the range of proteins found in this compartment and the types of modifications to which they are subject. For instance, while protein turnover is one of the most thoroughly investigated functions of the yeast vacuole, and many of the proteins that participate have been known for some time, this and related processes have yet to be explored at the systems level through the application of global proteomics approaches.

A lack of systems level knowledge of the range of proteins to be found in this organelle could mean there are luminal proteins that remain to be discovered, which in turn might pose an impasse for the rational analysis of many cellular processes and ultimately cellular engineering. If vacuolate cells, for instance those of yeast, are to be manipulated for enhanced nutritional quality, the provision of pharmaceuticals or their precursors, precursors for manufacturing purposes, or for environmental remediation applications, it will by critical that the research community has access to a virtual vacuole (‘vacuomics’) toolbox detailing the protein profile of the vacuole lumen and how the latter is established and maintained by intravacuolar reactions and transport across the vacuolar membrane. S. cerevisiae possesses only a moderate number of open reading frames, most of which are devoid of introns, and as one of the most molecularly manipulable genomically characterized eukaryotes, is a model system for the identification and definition of eukaryotic protein functions. Moreover, since it is vacuolate and the core machinery for protein delivery into and processing within the vacuole is likely conserved in other vacuolysosomal structures, investigations of S. cerevisiae have the potential to contribute to our understanding of not only other fungal systems and plants but also the lysosomal compartments of animal cells.

Using high-purity ‘proteomics-grade’ intact yeast vacuoles, 360 luminal polypeptide species have been resolved reproducibly by two-dimensional gel electrophoresis . Of these, 117 have been identified by MALDI-TOF MS and/or LCQ-MS through the deployment of ProteinLynx-, MASCOT- and/or SEQUEST-based protein sequence database searches in combination with Mr and pI considerations. The polypeptides identified, many of which correspond to alternate isoelectric and size states of the same parent translation product, can be assigned to 66 unique reading frames. In strict agreement with a predominantly lysosomal function for the yeast vacuole, most of the proteins identified are either canonical vacuolar proteases or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress that have entered this compartment for salvage purposes.

Roy and Diana Vagelos Program in Life Sciences & Management

On the surface, bioscience and business may seem like unrelated fields. But if the full benefits of science are to be realized, discoveries made at the laboratory bench must be taken to market and made accessible to society at large—a process demanding great skill both scientifically and managerially. Given the many recent advances in bioscience and biotechnology, never before has the need been so great for decision makers who can understand and advance scientific innovations as well as manage and promote them. It is with this in mind that the University of Pennsylvania launched the Vagelos Life Sciences & Management (LSM) program.

LSM is administered jointly between Penn's College of Arts & Sciences and the Wharton School. Each year, this undergraduate program enrolls approximately 25 exceptional students and offers them the opportunity to pursue an interdisciplinary curriculum combining bioscience and business, leading to the completion of two degrees: a Bachelor of Arts in a life science major, as well as a Bachelor of Science in Economics. LSM provides an ideal preparation for careers in the rapidly growing life sciences sector. The LSM program is suited to students with interests in health care; biomedical, agricultural, and environmental research and development; public policy; and the financial and strategic management of biotech enterprises.

Our LSM-related secondary research at the interface of life sciences and its implementation focuses on case studies that highlight the difficult transition from discovery in the laboratory to success in the market and/or toward the expansion of humanitarian efforts.  Examples of such case studies are “Statins: From Fungus to Pharma”  and “Ivermectin and River Blindness”, two feature articles aimed primarily at the educated lay reader.