Robert E. Hughes

A protein interaction network of the malaria parasite Plasmodium falciparum

Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein–protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen.

Systematic and large-scale two-hybrid screens

The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.

Calcium Binding, but Not a Calcium-Myristoyl Switch, Controls the Ability of Guanylyl Cyclase-activating Protein GCAP-2 to Regulate Photoreceptor Guanylyl Cyclase

Guanylyl cyclase-activating protein 2 (GCAP-2) is a recoverin-like calcium-binding protein that regulates photoreceptor guanylyl cyclase (RetGC) (Dizhoor, A. M., and Hurley, J. B. (1996)J. Biol. Chem. 271, 19346–19350). It was reported that myristoylation of a related protein, GCAP-1, was critical for its affinity for RetGC (Frins, S., Bonigk, W., Muller, F., Kellner, R., and Koch, K.-W. (1996) J. Biol. Chem. 271, 8022–8027). We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca2+-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca2+ than at high Ca2+ concentrations. We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs. Myristoylated or nonacylated GCAP-2 forms were expressed inEscherichia coli. Wild-type GCAP-2 and the Gly2→ Ala2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells. We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca2+- and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC. The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC. Nor does it affect the ability of the Ca2+-loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca2+ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.

Altered transcription in yeast expressing expanded polyglutamine

Expanded polyglutamine tracts are responsible for at least eight fatal neurodegenerative diseases. In mouse models, proteins with expanded polyglutamine cause transcriptional dysregulation before onset of symptoms, suggesting that this dysregulation may be an early event in polyglutamine pathogenesis. Transcriptional dysregulation and cellular toxicity may be due to interaction between expanded polyglutamine and the histone acetyltransferase CREB-binding protein. To determine whether polyglutamine-mediated transcriptional dysregulation occurs in yeast, we expressed polyglutamine tracts in Saccharomyces cerevisiae. Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines, and these profiles were compared to existing profiles of mutant yeast strains. Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyglutamine in either the nucleus or cytoplasm. Transcriptional repression was most prominent in yeast expressing nuclear expanded polyglutamine and was similar to profiles of yeast strains deleted for components of the histone acetyltransferase complex Spt/Ada/Gcn5 acetyltransferase (SAGA). The promoter from one affected gene (PHO84) was repressed by expanded polyglutamine in a reporter gene assay, and this effect was mitigated by the histone deacetylase inhibitor, Trichostatin A. Consistent with an effect on SAGA, nuclear expanded polyglutamine enhanced the toxicity of a deletion in the SAGA component SPT3. Thus, an early component of polyglutamine toxicity, transcriptional dysregulation, is conserved in yeast and is pharmacologically antagonized by a histone deacetylase inhibitor. These results suggest a therapeutic approach for treatment of polyglutamine diseases and provide the potential for yeast-based screens for agents that reverse polyglutamine toxicity.

Matrix Metalloproteinases are Modifiers of Huntingtin Proteolysis and Toxicity in Huntington’s Disease

Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntingtons disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the protease families-caspases and calpains. Identifying critical proteases involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest N-terminal polyglutamine-containing Htt fragment. We screened 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP-10, directly cleaves Htt and prevents cell death when knocked down in striatal Hdh(111Q/111Q) cells. Correspondingly, MMPs are activated in HD mouse models, and loss of function of Drosophila homologs of MMPs suppresses Htt-induced neuronal dysfunction in vivo.

Suppression of Neurodegeneration and Increased Neurotransmission Caused by Expanded Full-Length Huntingtin Accumulating in the Cytoplasm

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N terminus of the huntingtin (htt) protein. Here we describe the generation and characterization of a full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded htt is imported into the nucleus in detectable amounts. We find that expanded full-length htt (128Qhtt(FL)) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128Qhtt(FL) animals. Partial loss of function in synaptic transmission (syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length htt during early stages of pathogenesis.

Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan

While it is generally recognized that misfolding of specific proteins can cause late‐onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry‐based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)‐insoluble conformations during aging in Caenorhabditis elegans. SDS‐insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS‐insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS‐insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

A Human Protein Interaction Network Shows Conservation of Aging Processes between Human and Invertebrate Species

We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

Glucocorticoids suppress selected components of the senescence-associated secretory phenotype.

Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors, and proteases that can alter the tissue microenvironment and may promote age‐related pathology. To identify small molecules that suppress the senescence‐associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are approved for human use. Among the promising library constituents was the glucocorticoid corticosterone. Both corticosterone and the related glucocorticoid cortisol decreased the production and secretion of selected SASP components, including several pro‐inflammatory cytokines. Importantly, the glucocorticoids suppressed the SASP without reverting the tumor suppressive growth arrest and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Suppression of the prototypical SASP component IL‐6 required the glucocorticoid receptor, which, in the presence of ligand, inhibited IL‐1α signaling and NF‐κB transactivation activity. Accordingly, co‐treatments combining glucocorticoids with the glucocorticoid antagonist RU‐486 or recombinant IL‐1α efficiently reestablished NF‐κB transcriptional activity and IL‐6 secretion. Our findings demonstrate feasibility of screening for compounds that inhibit the effects of senescent cells. They further show that glucocorticoids inhibit selected components of the SASP and suggest that corticosterone and cortisol, two FDA‐approved drugs, might exert their effects in part by suppressing senescence‐associated inflammation.

Therapeutic opportunities in polyglutamine disease

Polyglutamine diseases comprise a class of familial neurodegenerative disorders caused by expression of proteins containing expanded polyglutamine tracts. Great progress has been made in elucidating the molecular mechanisms contributing to polyglutamine pathology, and in identifying potential drug targets. Although much remains to be learned, these advances provide an opportunity for rational approaches to target-based drug discovery.