Iris Lindberg - US grants

The biosynthesis of active opioid peptides, like the biosynthesis of other peptides and hormones, involves a sequential series of enzymatic steps begin with signal peptide cleavage and ending with various terminal modifications. Considerable information is available on the processing steps involved in the production of ACTH and beta endorphin from the opioid peptide precursor proopio- melanocortin; however, the processing pathways taken by peptides derived from the other two opioid peptide precursors, proenkephalin and prodynorphin, are not well understood. Since opioid peptides have been implicated in physiological processes ranging from blood pressure control to immunological function, it is important to understand the biosynthetic mechanisms responsible for enkephalin production. We have previously carried out studies on the biosynthesis of enkephalins in the adrenal medulla. We now propose to extend and complete these studies by establishing the sequence of posttranslational processing steps required for the production of the penta- to octapeptide enkephalins met-enkephalin, leu- enkephalin, met-enkephalin-arg-phe, and met-enkephalin-arg-gly- leu. We will rely heavily on the use of pulse- chase/immunoprecipitation techniques to carry out this work. We also propose to follow up our discovery of the phosphorylation of proenkephalin with studies on the regulation of this important posttranslational processing event, and explore the potential presence of other such modifications of enkephalin-derived peptides (such as sulfation and glycosylation). It is hoped that information gained through these studies will be of use in future investigations of opioid peptides within the nervous system, and will provide basis for the study of plasma enkephalins as hormonal regulators.

The production of enkephalins, like the biosynthesis of other hormones, involves a sequential series of enzymatic steps culminating in the liberation of the active opioid sequences. Opioid peptides have been implicated in physiological processes ranging from blood pressure control to immunological function; the biochemical basis for the addictive properties of drugs may also relate to deficiencies in endogeneous levels or in the turnover of opioid peptides. It is therefore important to gain a good understanding of the biochemical mechanisms involved in opioid peptide biosynthesis. My short-term goals are to define the sequence of cleavages of proenkephalin as well as to investigate the presence of posttranslational modifications. Other short-term goals relate to the study of a putative proenkephalin processing enzyme, a serine protease present in chromaffin granules. My long- term objectives are to understand how biologically active peptides are synthesized. Ultimately I would like to be able to study the cellular regulation of proenkephalin processing, and to use molecular biological techniques to investigate general questions relating to the specificity of hormone processing proteases. The RCDA, if awarded, will assist me in accomplishing the goals outlined above in two ways. Firstly, it will free me from Departmental obligations such as substantial teaching in service courses, enabling me to devote my complete attention to research throughout the year. Secondly, by securing this award, I will be exempted from service on additional committees, both at a Departmental and Institutional level. The Department of Biochemistry and Molecular Biology provides a supportive environment for the development of my research career. The newly-established peptide synthesis/sequencing facility will enable on-site synthesis of peptides required for the above studies. Resource personnel able to provide direction in the techniques of molecular cloning are also present. The fact that Department faculty receive considerable support from federal granting agencies means that expertise and equipment is readily available through active research programs in surrounding laboratories. In short, the environment at L.S.U. provides an extremely favorable milieu in which to carry out the studies described in this proposal. The award of an RCDA will greatly speed the rate at which these studies are accomplished.

Active opioid peptides like other neuropeptides and polypeptide hormones are formed through the action of intracellular processing enzymes which cleave and then modify precursor proteins. The long-term goal of these studies is to better understand the enzymatic mechanisms responsible for the proteolytic processing of proenkephalin to bioactive opioids, with emphasis on the role of the new subtilisin-like processing enzymes (PC1 and PC2) which have recently been described in neuroendocrine tissues. Since proteolytic cleavage represents the first step of the enkephalin biosynthetic pathway, it is likely that regulatory mechanisms which control opioid peptide production may involve these crucial proteolytic enzymes. This proposal represents our continued efforts to describe the proteolytic processing of proenkephalin by PC1 and PC2. Co-transfection experiments of enzyme cDNAs with proenkephalin cDNA will help us to better understand the interaction of opioid precursors with processing enzymes in a cellular context, while our overexpression experiments, which have provided us with purified recombinant proenkephalin, PC1 and proPC2, will provide information on the regulation of enzyme activity and the identity of in vitro products of proenkephalin digested with PCs. Anti sense blockade will be used to investigate a regulatory role for PC2 in the generation of active opioids. Site-directed mutagenesis will be employed to study catalytically important domains within PC1; this technique will also be used to investigate the biochemical basis for ordered processing of proenkephalin. Taken together, these experiments should provide significant progress toward our goal of understanding regulatory mechanisms in opioid peptide biosynthesis. Deficiencies in the biosynthetic capacity for opioid peptides may be responsible for the addictive properties of opiate drugs in certain individuals; thus the study of enzymatic mechanisms regulating endogenous opioid production is of extreme importance. A thorough understanding of regulatory mechanisms in opioid peptide synthesis might one day lead to enzyme-based drugs serving as therapeutic agents in opiate addiction.

DESCRIPTION (Applicant's Abstract): This renewal of a K02 award is requested in order to free the applicant from substantive teaching and administrative responsibilities to increase research productivity. During the past 4 years the PI has been able to devote more than 80% of her time to research activities, a level of effort which has resulted in a significantly enhanced ability to perform experiments, advise postdoctoral fellows and graduate students, and acquire new technologies both by collaboration as well as by hands-on experimentation. This enhancement of research effort is expected to continue during the renewal period of the application with the protection afforded by the K02 award; without it, teaching responsibilities are likely to increase dramatically, most notably by the assumption of course directorship in a major service course. The proposal is based upon two currently ongoing projects, one on the proteolytic processing of opioid peptide precursors, and one on the mechanism of action of the PC2 binding protein 7B2. The four specific aims are 1) to define the specificity of recombinant PC1 and PC2 using recombinant normal and mutant proenkephalins and related fluorogenic substrates; these experiments will delineate substrate preferences of the two PCs. 2) To identify novel PC1 inhibitors, study the biochemistry and cell biology of PC1 and PC2 inhibitors, and assess potential effects of targeted inhibitors on proenkephalin processing in vivo. These experiments will complement those described in the first specific aim as to enzyme specificity and should yield information on the design of synthetic enzyme inhibitors. 3) To determine the role of 7B2 in the regulation of proPC2 conversion in neuroendocrine cel1 lines, with a focus on the fate of the propeptide and the relationship of its cleavage/dissociation to that of 7B2 forms. 4) To explore the molecular interaction of 7B2 and PC2 through in vitro experiments. Taken together, these experiments should help us to understand the basic biochemistry of the prohormone convertases on opioid peptide precursors as well as the regulation of convertase activity.

DESCRIPTION (provided by applicant): The anthrax bacillus produces a three-component exotoxin of which an essential element for bioactivity is the protein known as protective antigen, or PA. PA binds to a cell surface receptor and is cleaved to generate a 63 kDa protein to which the one of the other anthrax toxins, LF and EF, can bind. Proteolysis and binding of PA permits internalization of a PA-LF complex into the cytosol, where it is able to attack cellular machinery, resulting in cell death. Since proteolytic cleavage of the PA anthrax toxin is obligatory for the manifestation of toxic activity, this cleavage step represents a natural target for pharmacologic intervention. Previous research has shown that this cleavage is performed by a cellular surface enzyme known as furin, a member of the family of eukaryotic subtilisins. This application is directed toward the idea that the cytotoxic action of anthrax toxin can be attenuated through inhibition of the activating cleavage event, resulting in lessened toxicity and cellular protection. In the last decade, several groups have shown that it is possible to block cleavage of bacterial toxins using engineered protein inhibitors of furin. We have recently used combinatorial chemistry techniques to identify a stable hexapeptide, D6R {D-hexa-arginine) which represents a potent, stable small molecule inhibitor of furin. Our preliminary data indicate that D6R can effectively inhibit furin-mediated cleavage of a bacterial toxin derived from Pseudomonas, blocking lethal effects in both cell lines as well as live animals. We here propose to systematically apply our studies of furin inhibition to the blockade of anthrax toxin activation. Specifically, we will investigate the use of D6R itself as a potential therapeutic in the attenuation of anthrax toxin cytotoxicity; examine the structural requirements for inhibition of furin-mediated cytotoxicity by D6R related molecules; and test D6R and/or other stable furin inhibitors identified in this work in animal models of anthrax toxicity. Our preliminary data showing potent inhibition of PA cytotoxicity by D6R support the idea that small molecule furin inhibitors will represent effective agents for the biologic attenuation of anthrax toxin cytotoxicity, the development of such antitoxin agents will add significantly to our ability to protect organisms against this pathogen.

The successful sequencing of the human genome has given rise to the next scientific opportunity of the twenty-first century: functional annotation of the proteome. About one-fifth of the genome encodes secretory proteins, a small number of which represent signaling molecules for G-protein coupled receptors (GPCRs). The major challenge that we will explore in this proposal is the comprehensive 'de-orphanization'(and thereby annotation) of the universe of peptides involved in neuronal GPCR signaling. There are currently about 100 non-olfactory orphan GPCRs, many of which are expected to use peptide ligands;however, fewer than a dozen novel peptides have been identified within the last eight years- and none at all within the last few years. Bioinformatics analyses indicate that the genome contains about 150 untested secretory proteins which possess biochemical similarities to known peptide precursors. We postulate that these proteins contain the missing peptide ligands for orphan GPCRs. However, in order to make these new orphan receptor- peptide matches, fresh approaches to peptide ligand identification are urgently needed. To identify novel peptide neurotransmitters we propose to take an innovative approach integrating expertise in bioactive peptide synthesis (Lindberg laboratory) with expertise in GPCR screening (Roth laboratory). We will experimentally confirm the presence of prohormone convertase-cleavable sites in a bioinformatically-derived list of putative precursors. Bioactive peptides will be generated from all validated precursors through large-scale in vitro posttranslational modification reactions using physiological enzymes (Lindberg laboratory). We will then discover cognate receptors to these peptides via functional screening against the entire genomic complement of known and orphan peptide receptors using facile screening technologies (Roth laboratory). Our results will enable us to match orphan receptors with novel peptide ligands, thus providing new neuropeptide-receptor signaling pairs. Since neuropeptide signaling pathways are critical to brain function and include pathways involved in mood and cognition, mental disorders, and drug reward, our results will significantly advance our understanding of mental health and disease, and may also generate new drug targets. While we will focus on obtaining and testing neuronally-expressed precursors/ligands and receptors, our research is also likely to uncover other ligand-receptor matches;thus a major impact on the many other physiological processes controlled by peptide- GPCR receptor signaling pathways is also anticipated.

DESCRIPTION (provided by applicant): One-fifth of the genome codes for secretory proteins;a small subset of these proteins represent peptide hormone signaling molecules. This proposal addresses the identification of novel secretory molecules involved in the control of metabolic function. Since the majority of neuroendocrine signaling molecules undergo proprotein convertase-mediated maturation, we plan to exploit the presence of genuinely cleavable sites in novel precursors to validate a set of potential peptide precursors. We will then test the bioactivity of correctly modified peptide products derived from these proteins. These assays will identify biologically active peptides acting on tissues that are key effectors of metabolic control: fat, liver and muscle. The proposed project represents a collaboration of the Lindberg laboratory with the secretory protein company Five Prime Therapeutics. Briefly, bioinformatics identification of large numbers of putative precursors by Five Prime will be followed by screening of HEK-expressed proteins with purified recombinant convertases (Lindberg laboratory). The information generated by the processing screen will then be used to direct large-scale recombinant protein production of promising precursors in the Lindberg laboratory. These purified precursors will be subjected to in vitro proteolytic and terminal maturation reactions in the Lindberg laboratory, and the resulting peptide products will then be tested in six metabolic assays targeted at fat, liver and muscle cell glucose metabolism at FivePrime Therapeutics. We expect that these studies will result in the identification of several novel peptide hormones contributing to metabolic control. These results should aid in our complete understanding of the hormonal control of glucose and lipid metabolism. PUBLIC HEALTH RELEVANCE: This is a discovery project which focuses on the identification of novel bioinformatics-identified peptide hormones. We will employ chemical processing of candidate precursors using physiological enzymes to make peptide mixtures which we will test in six different assays of glucose and lipid metabolism. We expect that these studies will result in the identification of several novel peptide hormones contributing to metabolic control.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) affects 5.2 million Americans over 65, a number expected to increase along with the general aging of the US population. While remarkable progress has been made in the last decade in defining the toxic effects of the amyloid species thought to be involved in loss of cognitive function, much remains to be learned regarding amyloid plaque pathogenesis. Recent data support the idea that chaperones, proteins which control folding homeostasis, contribute to proper neuronal function in a variety of ways. Our laboratory has recently shown that 7B2, a small secreted neuronal protein, is associated with brain amyloid plaques in tissues from both AD humans and from mouse AD models. These data are supported by five independent bioinformatics studies indicating that this protein represents a potential CSF biomarker for neurodegenerative disease. Although present in the secretory pathway and not the cytosol, 7B2 exhibits many biochemical characteristics similar to those of small heat shock protein chaperones. Like alpha crystallin, a member of the heat shock chaperone family, 7B2 can potently block the fibrillation of beta amyloid 1-42 in in vitro tests, and also blocks the cytotoxicity of beta amyloid added to Neuro 2A cell cultures. The current R21 proposal is to test the hypothesis that brain 7B2 levels can control the extent of amyloid plaque deposition and affect cognition in a known mouse model of Alzheimer's. We propose to cross existing mouse strains that either over- or underexpress 7B2 with the APP/PS1 Alzheimer's disease model mouse. Cognitive abilities will be assessed at 6 and 12 months of age in a Morris water maze. Plaque pathology will be quantitated in cortical and hippocampal slices in 6 animals of each genotype; 7B2 immunoreactivity will be measured in concert. Amyloid oligomerization state will be examined in brains of separate animals using chemical and centrifugal separation followed by ELISA and also correlated with genotype. Collectively, these experiments constitute a direct test of our hypothesis that 7B2 levels are negatively correlated with amyloid plaque and oligomer formation.

Demographic considerations predict an overwhelming burden on the U.S. health care system within the next few decades due to an influx of elderly patients with neurodegenerative disease. The most prevalent of these, Alzheimer's and Parkinson's diseases (AD and PD), are predicted to affect tens of millions of Americans by 2040. Both of these diseases involve progressively more aberrant brain proteostasis, associated with massive neuronal cell death. A variety of cytosolic and secreted brain chaperones contribute to maintenance of neuronal proteostasis in these and other neurodegenerative proteinopathies; of these, the secretory chaperone proSAAS has many compelling features. ProSAAS is expressed only in neurons and endocrine cells; because it traffics through the regulated secretory pathway, it becomes concentrated within dense core synaptic granules, and is released during neuronal activity. ProSAAS has been identified by five proteomics groups as a potential biomarker in neurodegenerative disease, and is found associated with aggregated proteins in the substantia nigra of PD patients as well as with amyloid plaques in AD?affected individuals. ProSAAS blocks the aggregation of both Abeta and alpha synuclein at highly substoichiometric ratios, and both endogenous overexpression as well as exogenous application reduce Abeta? and alpha synuclein?mediated neurotoxicity in primary neurons and cell lines. Most recently, we have shown that proSAAS overexpression is also functionally protective in vivo in a rat model of alpha?synuclein overexpression. In the proposed work, we will investigate the likely common mechanisms by which proSAAS protects neurons from neurotoxic aggregating proteins and peptides such as alpha synuclein and Abeta 1?42. We hypothesize that secreted proSAAS sequesters cytotoxic oligomers and fibrils extracellularly, reducing their concentrations at the synapse. Secondly, we hypothesize that endocytosed proSAAS acts intracellularly to similarly sequester cytotoxic proteins, speeding their degradation. Using cultured primary hippocampal and nigral neurons, we will determine whether proSAAS is involved in intracellular and extracellular Abeta and alpha synuclein sequestration. We will also determine whether intracellular expression of proSAAS confers a cytoprotective advantage compared to extracellular addition. Lastly we will assess whether endocytosed proSAAS accelerates the intracellular degradation of Abeta and alpha synuclein. In parallel, we will expand our exciting in vivo results to include the alpha?synuclein preformed fibril model to tease apart the potential sites of action of proSAAS in substantia nigra and striatum. Pre?degenerative changes in dopamine homeostasis, assessed using fast?scan?cyclic?voltammetry, will be correlated with proSAAS?mediated neuroprotection. Similarly, a mouse model of AD will be used to test the effects of proSAAS AAV?mediated over? and underexpression on the development of amyloid pathology. Collectively, these experiments will provide insight into biochemical mechanisms underlying the potent cytoprotective effects of the proSAAS chaperone protein.

SUMMARY In our original grant, we proposed to investigate the likely common mechanisms by which the proSAAS chaperone protects neurons from neurotoxic aggregating proteins and peptides, such as alpha synuclein and Abeta. We hypothesized that secreted proSAAS sequesters cytotoxic oligomers and fibrils extracellularly, reducing their concentrations at the synapse, and that endocytosed proSAAS might act intracellularly to similarly sequester cytotoxic species. Using cultured primary hippocampal and nigral neurons, we are currently investigating whether secretory proSAAS is involved in intracellular and extracellular Abeta and alpha synuclein sequestration, and whether secretory proSAAS also accelerates the intracellular degradation of Abeta and alpha synuclein. However, three major findings were made recently in carrying out the above project which take the work in an unexpected but important new direction. The first is the discovery that delivery of proSAAS to the cytoplasm, by expression of signal-less proSAAS (?cyto-proSAAS?), results in the liquid-liquid phase separation and formation of large (2 - 4 µm) symmetric proSAAS spheres, formed by dynamic fusion of smaller spheres. The second major finding is that these cyto-proSAAS spheres specifically interact with TDP-43216-414 aggregates, and efficiently sequester these aggregates within the sphere cores. Thirdly, and most importantly, a collaboration with the Shorter laboratory provided important information that the interaction between proSAAS and TDP-43 is cytoprotective in a yeast model cell system. The proposed supplement to our existing ?Common Mechanisms? grant is designed to determine whether cytoplasmic proSAAS should also be studied, not only in proteostatic mechanisms in Alzheimer's disease, but also in the context of TDP-43 aggregation in another neurodegenerative disease, fronto-temporal dementia. Obtaining a one-year supplement to investigate the functional properties of cyto-proSAAS will provide us with the opportunity to exploit our exciting findings regarding the highly unusual physical properties of cyto-proSAAS in forming ?aggregate sequestration? spheres. This supplement will also permit us to determine whether cyto-proSAAS expression is relevant to blocking Abeta and TDP-43 cytotoxicity in human cells (indeed, cyto-proSAAS expression may represent an improved avenue to achieve our original specific aim of ameliorating Abeta cytotoxicity -original Proposal Aims 1 and 3). Ultimately, this research will provide insight into whether proSAAS-mediated cytoplasmic aggregate sequestration should be further explored as a possible therapeutic approach in neurodegenerative disease. Lastly, it should be mentioned that given the unpredicted costs of the Covid-19 research shutdown, we clearly require additional funding to work on cyto-proSAAS, as we will otherwise be solely dedicated to recovering lost time in completing our original Aims. 1