1997 — 2000 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding B Sheet Structure in Aqueous Solution @ Scripps Research Institute
DESCRIPTION: This is a competitive renewal request for continuation of a program of studies aimed at understanding the chemical principals governing the folding, stability and self-association of beta-sheet structures in aqueous solution. Three lines of investigation are proposed: 1) the design and synthesis of templates that can be inserted into sequences to facilitate sheet formation and to determine of what kind of amino acids are thermodynamically favored to form sheets, and the role of hydrophobic clusters and hydrogen bonds in driving sheet formation; 2) studies of a naturally occurring 38-residue, 3-stranded antiparallel beta-sheet domain (termed the WW domain because of its 2 Trp residues) aimed at understanding the interactions that stabilize the domain structure; 3) studies of intermolecular beta-sheet formation using hosts that can selectively bind peptides. The peptides are obtained by solid phase chemical synthesis, and in the case of the WW domain, using a bacterial expression system. The peptide structures are evaluated by analytical ultracentrifugation, CD, FTIR, NMR, mass spectroscopy, binding of hydrophobic probes (ANS), fluorescence, as well as sequence analysis, in the case of the WW domain motif that has been identified in a number of proteins.
|
1 |
1998 — 2005 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Probing the Biochemical Mechanism of Amyloid Diseases @ Scripps Research Institute
DESCRIPTION: This is the first competitive renewal following R29 funding to investigate the mechanisms of amyloid disease. The long term goals of this research program are to understand the biochemical mechanism of human amyloid disease and develop new therapeutic strategies to test hypotheses about the etiology of these diseases. The common features of human amyloid disease involve the extracellular deposition of proteins in a fiber morphology. Significant circumstantial evidence suggests that fibrils directly cause the neuropathology of the brain associated diseases and this concept has been generalized as the "amyloid hypothesis". This outstanding young investigator focuses his proposal on two amyloid diseases that do not directly involve the brain. These are familial amyloid polyneuropathy and senile systemic amyloidosis. In the familial disease, a mutated form transthyretin (prealbumin) (TTR) is deposited as fibrils in peripheral nerves or in specific organs while in the senile form of the disease normal TTR forms primarily in cardiac tissue. These diseases were chosen in order to test the general amyloid hypothesis by utilizing small molecules that can inhibit TTR amyloid fibril formation to directly assess whether inhibition of transthyretin fibril formation is sufficient to prevent the onset of amyloid disease. The first specific aim of this proposal is centered around further testing the conformational change hypothesis, which suggests that tertiary structural changes are involved to make a given protein amyloidogenic. From prior work, it was shown that TTR amyloid fibril formation results from the self-assembly of an alternative tertiary structure of the protein. This result will be extended with continued funding to apply numerous biophysical methods including mass spectrometry and NMR to precisely determine the structure of the wild type amyloidogenic intermediate, and to compare this structure to the familial amyloid polyneuropathy associated variants of transthyretin (TTR). Previous work during the past funding period has shown that these TTR variants are much less stable and denature a thousand fold faster than wild-type TTR. Under continued support, Dr. Kelly will investigate tetramers composed of mixed wild-type and familial variants to assess their stability, rates of pH mediated denaturation and amyloidogenicity. The second aim involves a structure-based drug design strategy utilizing the synergistic application of x-ray crystallography, organic synthesis and parallel screening in order to identify high affinity binding amyloid inhibitors that stabilize the native state of TTR and prevent the pH mediated conformational changes that result in fibril formation.
|
1 |
2000 |
Kelly, Jeffery W |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Acquisition of a Mass Spectrometry Resource @ Scripps Research Institute
The research programs in multiple laboratories at Scripps have rapidly developed in directions which require extensive hands-on access to a mass spectrometer for widely varying types of experiments. We propose the acquisition of a Thermoquest LCQ quadrapole ion trap mass spectrometer with a MAGIC HPLC attachment to address this critical need. The new instrument will be the cornerstone of a newly established User Access Mass spectrometry facility. This new facility, including the proposed LCQ and a MALDI instrument acquired through other sources, will be supervised by a dedicated Ph.D. level scientist. The supervisor will be responsible for administration of the facility, training of users, and assistance with design and implementation of mass spectrometry experiments. The LCQ will be an indispensible analytical tool for the identification and characterization of proteins, for identification of post-translational modifications, for total chemical synthesis of proteins, for the discovery of small molecule inhibitors of amyloid formation, and for analysis of combinatorial compound libraries. The proposed LC-MS system, with MST capabilities, will provide a wide variety of experimental applications. The SEQUEST software package will be obtained to analyze protein mass spectrometric data and search the protein databases. This equipment will allow separation of proteins and peptides by HPLC, direct analysis of their molecular weight by electrospray mass spectrometry, and in addition, will allow tandem mass spectrometry to obtain sequence information. The direct user-access of this instrument will enable graduate students and postdocs to perform experiments that are beyond what can be achieved in a routine fee-for-service facility. Scientists will be able to tailor MS experiments to their specialized applications, in a format where the experiments can be optimized with direct input from the user. The LCQ LC-MS system should prove to be a powerful research tool that provides critical analytical information to a wide variety of scientists in a wide variety of disciplines.
|
1 |
2001 — 2003 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Peptides to Imidates and Back-Towards Bioavailibility @ Scripps Research Institute
The goal of this research is to develop improved chemistry to transform simple peptides into products lacking or with reduced numbers of amide functional groups, in some cases temporarily. The chemistry we aim to develop has its origins in biosynthesis, wherein enzymes activate amide functional groups towards nucleophilic attack by various side chains leading to the formal intramolecular cyclodehydration of the participating amide resulting in oxazoline and thiazoline ring formation. One third of this proposal will focus on developing synthetic methodology to convert peptides into heterocycles irreversibly with retention of Calpha and Cbeta stereochemistry. The availability of a wide variety of alpha- and beta-amino acids allows the synthesis of peptides tailored to produce the desired imidates, thiomidates and related structures. A variety of side chain protected and unprotected peptides will be synthesized to scrutinize the efficiency, as well as the regio- and stereoselectivity of the formation of a spectrum of heterocycles. The second portion of this proposal will focus on developing methodology to reversibly mask amide functional groups in peptides to make them bioavailable. Amide bonds will be converted to imidate esters, or the like, with favorable membrane translocation properties to facilitate cellular and/or oral bioavailability, where subsequent hydrolysis regenerates the peptide. The membrane translocation properties of neutral imidate, thioimidate or similar backbones should be dramatically improved as a result of the reduction in the number of hydrogen bond donors and acceptors, which correlates with peptide membrane translocation ability. The cationic masked amides aim to take advantage of a newly discovered active transport system to mediate membrane translocation. Making peptides and proteins generally bioavailable is the long term goal of this specific aim. The remaining specific aim will focus on evaluating the biological activity of the heterocyclic and acyclic imidate and thioimidate products produced in this project. We will concentrate on antibacterial activity, particularly towards resistant strains, RNA binding, and antitumor activity. In the first and the third cases, these compounds will be submitted to screens at Novartis and the National Cancer Institute, respectively. In the second case, fused heterocyclic libraries will be prepared and their interactions with RNA targets evaluated by our Scripps collaborator Jamie Williamson.
|
1 |
2002 — 2019 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding Beta-Sheet Structure in Aqueous Solution @ Scripps Research Institute
DESCRIPTION (provided by applicant): The long term objective of this research is to understand the structural principles of beta-sheet folding using a small protein that folds by a two state mechanism. We utilize sequences of the Pin WW domain, a 34 residue 3-stranded antiparallel Beta-sheet, incorporating both natural and unnatural amino acid mutations to understand the structural features that are critical for the transition state formation and ground state stability. Our ability to chemically synthesize WW domains with varied backbone connectivity and a constant Beta-sheet core structure allows us to understand what aspect of topology (structure) is important for predicting folding rates-a prediction that is accurate within an order of magnitude or so. Synthetic accessibility also allows us to make subtle changes in WW domain structure to better understand how these changes influence the range of folding rates exhibited by WW domain variants. Thermodynamic and kinetic data for mutations at nearly every position in the sequence can be processed to afford a Phi (phi) analysis (phi) = deltadeltaG++/deltadeltaG) to discern the extent to which a perturbation in the free energy of folding is mirrored in the transition state. We make predictions about the importance of certain structural features including hydrogen bonding, hydrophobic interactions, conformational preferences, chain connectivity (topology), etc. in both the ground state and transition states that can be tested experimentally using a phi analysis. Understanding Beta-sheet folding is important to improve fold predictions from sequence and to begin to understand the balance between 13-sheet folding and misfolding-the latter process affords aggregates that appear to cause neurodegeneration.
|
1 |
2002 — 2006 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Multidisciplinary Analysis of Gelsolin Amyloid Disease @ Scripps Research Institute
DESCRIPTION (provided by applicant): The six-domain protein gelsolin functions in plasma (extracellular isoform) to disassemble actin filaments as part of the actin scavenging system. The D1 87N or Y mutation in domain 2 facilitates two aberrant proteolytic cleavages affording a 71 residue fragment that is found in amyloid fibrils in humans, putatively causing Familial Amyloidosis of Finnish type (FAF). We have discovered that the protease furin (found in the Golgi and endocytic compartments) and, potentially, related members of the prohormone convertase family effect the initial cleavage. This result allows us to probe the mechanism of this disease in an efficient fashion and provides a clear approach to intervene with small molecules. The second cleavage occurs at an unknown cellular/extracellular location. We have set out to understand the role of the FAF mutations in amyloidogenicity using biophysical studies in vitro (Aim 1) under conditions found in the trans-Golgi (pH 6.5, 200 uM Ca+2) in combination with cell biological studies focusing on both the export and import of gelsolin and fragments thereof in Aim 2, as well as transgenic animal studies in Aim 3. The goals of Aim 1 are to carry out a thermodynamic stability assessment on several different gelsolin constructs in addition to proteolysis sensitivity studies and structural comparisons of the WT and FAF variants to understand how these mutations lead to furin proteolysis. Small molecule inhibitors of the initial 172-173 cleavage will be sought using a cell-based screen. We will also carry out mechanistic and structural studies to better understand gelsolin fibril formation. In Aim 2, we will use a variety of biochemical, molecular and morphological approaches to further explore the site of processing of mutant gelsolin by furin and potentially other members of the prohormone convertase family. Moreover, we propose to identify the protease(s) involved in the second cleavage step leading to generation of the amyloidogenic fragment. In Aim 3, two transgenic animal models of FAF will be produced, one having one copy of WT and one copy of D187N plasma gelsolin (+/D187N) and the other having one copy of D187N on the knockout background (-/D187N) to improve our understanding of the relationship between aberrant processing, fibril formation and neurodegeneration. These mice provide a means of testing the small molecule therapeutics that come from Aims 1 and 2. Our long-term goal is to test the amyloid hypothesis in pathology by linking the cell biology and biophysical data to studies on transgenic animals and small molecule inhibitors that prevent misfolding and/or improper processing.
|
1 |
2005 |
Kelly, Jeffery W |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Characterization of Intermediate Species in Aggregation of Amyloid Beta Peptide @ Scripps Research Institute |
1 |
2005 — 2013 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Metabolite Initiated Protein Misfolding and Amyloidosis @ Scripps Research Institute
DESCRIPTION (provided by applicant): Degenerative diseases are associated with tissue-localized aggregation of proteins in a variety of morphologies, including fibrillar cross-¿-sheet assemblies referred to as amyloid fibrils. The most prominent neurodegenerative diseases among the elderly, Alzheimer's disease, and the Lewy body diseases (including Parkinson's disease) appear to result from a loss in the balance between protein synthesis, folding to a native functional state or maintenance of the natively unfolded state, aggregation, and degradation. In Alzheimer's disease, the natively unfolded A¿ peptide forms fibrillar aggregates that are the principal component of intracellular and extracellular plaques, along with intracellular deposits of hyperphosphorylated tau. Parkinson's disease is characterized by cytoplasmic a-synuclein aggregates associated with degenerating dopaminergic neurons in the substantia nigra and other brain areas. Clinical observations suggest a correlation between oxidative stress/inflammation, aggregation and degenerative diseases. Past studies have demonstrated that hydrophobic aldehydes derived from oxidative stress, in particular 4-hydroxynonenal and the atheronals, exacerbate aggregation through thermodynamic and kinetic perturbations mediated by covalent and non-covalent modifications of A¿ and a-synuclein, which may help to explain the occurrence of sporadic Alzheimer's and Parkinson's disease. The research proposed herein seeks to understand the effect of hydrophobic aldehydes on the maintenance of protein homeostasis in cells and multicellular organisms and the effect that these covalent modifications have on degenerative disease phenotypes in organismal models of degenerative diseases. Besides evaluating the influence of a new set of common lipid-derived aldehydes on aggregation in the absence and presence of biologically relevant membranes, we will discern their ability to inhibit the biological machinery that maintains organismal protein homeostasis, including the disaggregase activity recently discovered in human cells. We propose new methodology to follow protein aggregation in living human cells and in multicellular organisms using FlAsH fluorophores, to complement a previously developed kinetic aggregation assay in which seeding of aggregation by cell homogenates is the readout. Using these methods, we will assess the influence of the hydrophobic aldehydes on protein aggregation in vivo and discern whether quantification of aggregation in organisms explains toxicity as a function of aldehyde concentration in organisms with low or high levels of aggregation-prone protein expression. Transcriptional analysis of the protein homeostasis network upon aldehyde treatment in the presence of controllable levels of aggregation-prone proteins should provide much insight into the etiology of sporadic neurodegenerative diseases associated with aging, aggregation, and oxidative stress. PUBLIC HEALTH RELEVANCE: Clinical observations suggest a correlation between oxidative stress/inflammation, aggregation and degenerative diseases, such as Alzheimer's disease and Parkinson's disease. This project seeks to understand the influence of protein-modifying hydrophobic aldehydes, derived from the aberrant oxidation of membrane components, on the maintenance of protein homeostasis in cells and multicellular organisms, and the effect that these covalent modifications have on degenerative diseases. We will explore the hypothesis that covalent modifications of aggregation-prone proteins in organisms could exacerbate aggregation and the associated pathology and possibly also modify and inhibit the biological machinery that maintains cellular protein homeostasis.
|
1 |
2006 — 2014 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding and Ameliorating Loss-of-Function Protein Misfolding Diseases @ Scripps Research Institute
DESCRIPTION (provided by applicant): Many lysosomal storage disease-associated mutant enzymes exhibit compromised endoplasmic reticulum (ER) folding and therefore are subjected to degradation instead of being trafficked to the lysosome, where they normally degrade their substrates. This leads to lysosomal substrate accumulation and thus, pathology. In Specific Aim 1 we aspire to identify proteostasis network components responsible for (1) folding wild type and Gaucher disease-associated mutant ?-glucocerebrosidases (GCs) in the ER, (2) trafficking them through the Golgi and on to the lysosome, (3) stabilizing/activating them in the lysosome, and (4) degrading misfolded GC in the ER by immunoisolating the GC interactome (antibody recognizes the extreme C-terminus and is thus conformation and mutant insensitive) and identifying the proteins by mass spectrometry. Mutant ?- glucocerebrosidases exhibit overlapping interactomes with distinct members because they accumulate in various subcellular compartments. GC interacting partners, prioritized on the basis of 4 methods, will be RNAi depleted in L444P GC cells to discern what extent GC proteostasis is perturbed using the intact cell GC activity assay, endo H sensitivity and immunofluorescence colocalization. Several novel GC proteostasis network components have already been identified, allowing several hypotheses regarding the GC proteostasis network to be tested. Simply lowering the growth temperature of patient-derived cells to 300C, inducing the unfolded protein response (UPR), or increasing ER Ca2+ concentration with ryanodine antagonists enhances mutant enzyme folding, trafficking and function enabling the interactome of adapted cells vs. controls to be determined to test numerous hypotheses about mechanism, including whether these results extend to other lysosomal storage diseases. In Specific Aim 2, we explore which genetically-encodable, small molecule regulated UPR- associated transcription factors individually and in combination can enhance mutant lysosomal enzyme folding, trafficking and function. Understanding the transcriptome upregulated by these transcription factors and combinations affording heterodimeric transcription factors cross referenced to the upregulated proteome in proteostasis network adapted cells will provide additional mechanistic insight into the enhancement in proteostasis. Unregulated overexpression of XBP1s restores L444P GC proteostasis motivating the development of aryl oxime ether IRE1 activators that will be tested in patient-derived cells from multiple lysosomal storage diseases to discern efficacy. IRE1 Inhibitors should be useful to cancer researchers. PUBLIC HEALTH RELEVANCE: This research defines the protein homeostasis network that can be altered to enhance mutant lysosomal storage disease-associated enzyme folding, trafficking and function. Adapting the protein homeostasis network with small molecules offers the possibility of ameliorating multiple lysosomal storage diseases of similar etiology by fixing the folding and trafficking of the mutant enzyme instead of replacing the enzyme-the current standard of care, which is expensive, disease specific and not useful for alleviating neuropathic lysosomal storage diseases. Small molecule adaptors of cellular protein homeostasis used in combination with pharmacologic chaperones that bind to and stabilize a particular protein are envisioned to be a very promising future approach for treating loss-of-function diseases, of which the lysosomal storage diseases of focus herein are one class.
|
1 |
2006 — 2021 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Probing the Biochemical Mechanism of Amyloid Disease @ Scripps Research Institute
We seek to understand how the process of transthyretin (TTR) aggregation, and which aggregate structures lead to the dysfunction and ultimately the death of post-mitotic tissue in the TTR amyloid diseases. Understanding this structure-proteotoxicity relationship would enable the development of novel therapeutic strategies, and the establishment of early diagnostic biomarkers that quantify response to therapy. A mutation in one TTR allele results in destabilized heterotetramers comprising mutant and wild type (WT) TTR subunits being secreted from the liver, which exhibit faster rate-limiting tetramer dissociation and monomer misfolding, affording a spectrum of aggregate structures, including amyloid fibrils linked to autosomal dominant familial TTR amyloid cardiomyopathy. There is also sporadic WT TTR cardiomyopathy, affecting as much as 10% of the elderly male population. In a clinical trial assessing the potential of tafamidis (a drug that binds to and stabilizes the native TTR tetramer preventing misfolding and aggregation of newly synthesized TTR) for treating TTR cardiomyopathy, the amyloid load in the heart did not detectably change on the timescale of the tafamidis clinical response, rendering it unlikely that amyloid infiltration of the heart is the main driver of mortality. In Specific Aim 1, we will take a fresh look at the pathology of the human heart using the CLARITY method to probe the 3D relationships between the non-native TTR structures present, the host molecules to which the aggregates are colocalized, discern whether aggregates are inside or outside heart cells, characterize infiltration by immune cells such as macrophages, and probe whether there are any discernable autonomic nervous system deficiencies. Another key question is why does WT TTR aggregate? We will test the hypothesis that a minor population of a less kinetically stable, alternatively folded TTR tetramer forms during non-optimal cellular folding or is produced during failed lysosomal degradation, affording a TTR tetramer that is unstable and leads to aggregation. In Specific Aim 2, we will use antibodies and peptide probes to isolate non-native TTR from polyneuropathy patient plasma to characterize the circulating non- native structure(s) by atomic force microscopy and cryo-electron microscopy in collaboration with the Lander Lab, to test the hypothesis that distinct aggregate structures drive tissue tropism. We will compare the structure of aggregates from V30M plasma (a pure polyneuropathy) vs aggregates from a pure cardiomyopathy to assess their relative cytotoxicities to DRG neurons vs cardiomyocytes, to test the idea that the tissue tropism of these diseases is aggregate structure-based. With the Coelho group, we will continue to improve early polyneuropathy diagnosis by studying V30M carriers as they progress to polyneuropathy patients using unbiased plasma proteomics. Proteomics will also be used to attempt to understand why ? 30% of TTR polyneuropathy patients do not respond to any approved therapies, focusing on inflammatory and immune-modulatory molecules and cells that may become drivers of polyneuropathy.
|
1 |
2007 — 2010 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding Beta-Sheet Structure in Aqueous Solution. @ Scripps Research Institute
DESCRIPTION (provided by applicant): The long-term goal of this project is to understand beta-sheet folding energetics at a level that enables protein and proteomimetic design for therapeutic purposes. Comprehending beta-sheet folding energetics and contrasting them with misfolding energetics is essential for understanding the role of protein folding in health and disease, and for designing backbone modified proteins with enhanced physical properties. In specific aim 1 (50% effort), we employ a backbone and side chain mutagenesis strategy to test the hypothesis that a few energetically key residues make context-dependent, synergistic, hydrophobic and H-bonding contributions to native state stability. Such residues may be identifiable a priori owing to their extent of solvent exposure. Furthermore, we test the idea that backbone amide transfer free energies may be sensitive to the size and type of flanking side chains;if so this feature can easily be factored into beta-sheet design. In addition, we will explore the possibility of replacing pairs of solvent shielded backbone amides that make stabilizing H-bonds with E-olefin dipeptide isosteres to discern whether a backbone- backbone hydrophobic interaction could replace one or more H-bonds. This is the first step towards designing and synthesizing proteomimetics that fold and function despite having significantly fewer amide bonds than their ribosomally derived counterparts. Such mimetics could have substantially better membrane permeability properties than standard proteins, perhaps enabling aerosol and oral bioavailability. In specific aim 2 (50% effort), we take on the challenging task of understanding how N-glycosylation influences beta-sheet folding energetics, so that we can integrate this information into protein folding and secretion models that also consider the role of chaperones, folding enzymes, and degradation machinery. Any factor that influences protein folding kinetics and thermodynamics is likely to affect the partitioning of proteins between degradation pathways and normal folding and trafficking pathways. We test several hypotheses, including the idea that N-glycosylation of asparagine residues influences beta-sheet folding kinetics and thermodynamics, that a phi-value analysis as a function of oligosaccharide structure will reveal molecular details of the mechanism by which N-glycosylation influences folding, and that multiple glycosylation sites collaborate to influence folding of immunoglobulin domains, the fold found in antibody drugs.
|
1 |
2007 — 2010 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Multidisciplinary Analysis of Gelsolin Amyloid Disease @ Scripps Research Institute
DESCRIPTION (provided by applicant): Plasma gelsolin (PG), the extracellular isoform of a six domain protein that has an intracellular counterpart, functions in blood to disassemble actin filaments as part of the actin scavenging system. D187 mutations in PG render domain 2 proteolytically labile and aggregation prone, leading to a gain of toxic function disease associated with the process of amyloidogenesis. We have discovered that the D187(N,Y) mutations prevent Ca2+ binding to domain 2, rendering plasma gelsolin susceptible to aberrant endoproteolysis by furin in the Golgi during secretion. Once outside the cell, the 68 kDa fragment is cleaved again by specific matrix metalloproteases (MMPs) including MT1-MMP (MMP14) affording 5 and 8 kDa fragments. The amyloidogenesis of these fragments is hastened by sulfated glycosaminoglycan components of the extracellular matrix (ECM) found in affected tissues. The overall goals of this work are to understand the amyloid pathological cascade and to develop an effective therapeutic strategy against the gelsolin amyloidoses. In Specific Aim 1, we will characterize the peptides comprising human amyloid to be sure we identify all MMPs involved, perform biophysical studies that will reveal the mechanism of gelsolin amyloidogenesis and elucidate how the oligosaccharides composing the ECM accelerate and possibly stabilize gelsolin amyloid. In Specific Aim 2, we will focus on developing and characterizing both cell-based and murine models of gelsolin familial amyloidosis (FAF). Cell-based models will allow us to characterize the trafficking pathways involved in FAF disease and understand compartmentalization of the pathological cascade. The FAF mouse model developed in the first funding period faithfully recapitulates many features of FAF pathology, including the intracellular inclusions also associated with inclusion body myositis (IBM), the most common muscle degenerative disease in the aging population. These valuable models will be used both to scrutinize our understanding of the FAF pathogenic cascade and to evaluate therapeutic strategies. In Specific aim 3, we will use the cell- and murine-based models of FAF to evaluate the potential of MMP inhibitors and small molecules that antagonize the ECM-gelsolin amyloid interaction to ameliorate FAF. The elucidation of the FAF pathogenic cascade together with the development of candidate pharmacologic agents will significantly advance our understanding of human amyloid diseases.
|
1 |
2009 — 2013 |
Kelly, Jeffery W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Mechanisms Linking Aging, Abeta Proteotoxicity and Neurodegeneration @ Scripps Research Institute
DESCRIPTION (provided by applicant): Aging increases the risk of neurodegeneration. Strong evidence implicates aggregation-mediated proteotoxicity as the cause of neurodegeneration in numerous clinically important diseases, including Alzheimer's disease, although the etiology is unclear. Emerging genetic data suggest that the aging process is linked by signaling pathways to the fidelity of protein homeostasis, including the ability to recover or dispose of misfolded or aggregated proteins. Overall this program project strives to meet two goals: 1) to understand the organismal, cell biological and molecular bases for the pathways that protect organisms from protein aggregation, and 2) to determine how these pathways become compromised as an organism ages. The Kelly Laboratory will focus on the biochemical characterization of the pathway(s) and the underlying molecular determinants of the disaggregase activity that appears to protect against age onset proteotoxicity in C. elegans and murine models of Alzheimer's and in human cells and they will test the hypothesis that amyloidogenesis is a constitutive process. The Balch Laboratory will generate senescence cell models to probe the role of age-dependent changes in exocytic and endocytic APR and Abeta processing that appear to contribute to proteotoxicity and will employ their expertise to perturb the exocytic and endocytic pathways to understand the genesis of Abeta proteotoxicity. The Dillin Laboratory will utilize genetic, proteomic and bioinformatics approaches and animal models of Alzheimer's disease to understand how and which aging-associated signaling pathways and downstream determinants affect proteotoxicity and they will carry out Abeta aggregate structure toxicity assessments in the worm Alzheimer's model. The bioinformatics, proteomics and neurosciences and neuropathology cores are each intimately associated with two or more of these projects that are themselves highly interdependent. The results obtained from this project will not only provide insight into the relationship between aging and neurodegeneration, but should provide the information necessary to develop therapeutic strategies for age-associated neurodegenerative diseases.
|
1 |
2009 — 2013 |
Kelly, Jeffery W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Identification of the Molecular Basis of Proteotoxicity Protection @ Scripps Research Institute
Aging increases the risk of neurodegeneration. Strong evidence implicates aggregation-mediated proteotoxicity as the cause of neurodegeneration in numerous clinically important diseases, including Alzheimer's disease, although the mechanism remains unclear. Emerging genetic data suggest that the aging process is linked by signalling pathways to the fidelity of protein folding, including the ability to recover or dispose of misfolded or aggregated proteins. Overall this program project strives to meet two goals. First, to understand the organismal, cell biological and molecular bases for the pathways that protect organisms from protein misfolding, and second, to determine how these pathways become compromised as an organism ages. The Kelly Laboratory will focus on four specific aims related to the key questions outlined directly above. These are: (1) to utilize biological and chemical approaches to identify and characterize the pathway(s) and the underlying molecular determinants of the fractionatable and separable disaggregation and proteolysis activities that appear to protect against age onset proteotoxicity in C. elegans models (in collaboration with the Dillin, Yates and Balch Laboratories);(2) to do the same with mouse AD models and mammalian cell lines, and to compare the results from these systems to the results obtained from C. elegans (also in collaboration with the Dillin, Yates and Balch Laboratories);(3) to employ biological and chemical approaches and especially immunoelectron microscopy to discern the subcellular location of Abeta aggregates in daf-2 RNAi or hsf-1 RNAi treated, Abeta-expressing worms, as well as to scrutinize the hypothesis that there is an active aggregation pathway or activity as implied from genetic data (in collaboration with the Balch, Dillin and Bannykh Laboratories), although other explanations for the data are possible;and (4) to test the hypothesis that amyloidogenesis is constitutive in the Borchelt Alzheimer's murine model long before Abeta fibrils are detectable immunohistochemically or behavioral phenotypes are observed, i.e., that amyloidogenesis happens as a consequence of the inherent inefficiencies in protein homeostasis (a subhypothesis is that constitutive amyloidogenesis only becomes toxic when the detoxification pathway(s) or activities drop below a threshold level due to organismal aging) (in collaboration with the Dillin, Balch, Bannykh and Masliah Laboratories).
|
1 |
2009 — 2010 |
Balch, William Edward [⬀] Dillin, Andrew G. Kelly, Jeffery W Morimoto, Richard I. (co-PI) [⬀] Wiseman, Rockland L (co-PI) [⬀] |
RC2Activity Code Description: To support high impact ideas that may lay the foundation for new fields of investigation; accelerate breakthroughs; stimulate early and applied research on cutting-edge technologies; foster new approaches to improve the interactions among multi- and interdisciplinary research teams; or, advance the research enterprise in a way that could stimulate future growth and investments and advance public health and health care delivery. This activity code could support either a specific research question or propose the creation of a unique infrastructure/resource designed to accelerate scientific progress in the future. |
Sensing Protein Folding Capacity in the Cell During Aging @ Scripps Research Institute
DESCRIPTION (provided by applicant): This GO grant addresses the critical need for development of biosensors that detect protein dysfunction during aging. The long-term health of all cells is inextricably linked to protein folding and sustainability of function. This is achieved by protein homeostatasis or 'proteostasis'(Balch et al. (2008) Science 319: 916), a complex network of molecular interactions that determines the health of the proteome. Proteostasis balances protein biosynthesis, folding, translocation, assembly/disassembly and clearance with the challenges imposed by environmental or physiological stress that results in a continual flux of misfolded and damaged proteins that the cell must manage. An imbalance, if left unattended can result in severe molecular damage to the cell, dysregulation of key tissues leading to pathology, and susceptibility to nearly all of diseases of aging. Adaptation and survival requires an ability to sense these damaged proteins and to coordinate induction of protective stress response pathways, chaperone and clearance networks. Despite the abundance and apparent capacity of the proteostasis network to restore the folding equilibrium, the cell appears to be poorly adapted for chronic proteotoxic stress as occurs when certain aggregation-prone proteins are expressed, for instance, in neurodegenerative aging diseases. We have hypothesized that this decline in repair activities, that challenges the integrity of the proteome, is influenced strongly by genes that control aging- thus linking stress biology, metabolism (diet), and protein homeostasis with health and human lifespan. The proposal brings together the complementary strengths of the Balch, Kelly and Wiseman laboratories at The Scripps Research Institute, the Dillin laboratory at the Salk Institute and the Morimoto laboratory at Northwestern University, to develop and test a new set of molecular tools that will globally report on the health of the proteome during aging. These groups form the Proteostasis Aging Sensor Consortium (PASC) to develop 'proteostasis sensors", innovative molecular reporters that will provide real- time assessment of the capabilities of protein folding quality control in each compartment of the cell, and in tissue and organismal models. These innovative probes will assess the consequences of protein damage, cell stress, aging and diseases of protein conformation that influence human longevity. The impact of these studies on the aging field is very broad and extends across all areas of biology and medicine. The combined collaborative efforts from the members of the PASC will leverage the tools, techniques and knowledge of protein homeostasis and aging to gauge the folding environment within cells and animals, and provide the next generation tools that will considerably accelerate efforts in the aging sciences. PUBLIC HEALTH RELEVANCE: The long-term health of mankind during aging is inextricably linked to protein folding and sustainability of protein function in spite of the many challenges imposed by environmental and/or physiological stress. Longevity requires an ability to sense damaged proteins and to coordinate induction of protective pathways and clearance networks responsive to genes that control aging, stress biology and metabolism (diet). This proposal by the Proteostasis Aging Sensor Consortium (PASC) consisting of the Balch-Dillin-Kelly-Morimoto- Wiseman groups will develop and test a new set of innovative molecular tools, referred to as proteostasis sensors that will globally report in real-time on the health of the human proteome during aging. The impact of these studies on the aging field is necessarily very broad and extends across all areas of biology and medicine related to human health.
|
1 |
2009 — 2013 |
Kelly, Jeffery W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative Core @ Scripps Research Institute
The objective of the Administrative Core (Core A) is to facilitate smooth and cost-effective communication, and to enhance collaborations amongst the Cores and Projects described in the Program Project Grant proposal. In this respect, we envision three Specific Aims for the Administrative Core. In Specific Aim 1, the Administrative core will facilitate communication between the Core and Project Leaders and all scientific and administrative personnel involved. This will be done through monthly meetings which will be archived by placing the PowerPoint presentations on the intranet website we have already created for this purpose. Other relevant materials will also be posted (e.g. all quarterly reports, manuscripts). A brief quarterly newsletter featuring the most important progress made will also be posted to the website and sent to all participants by e-mail. This Core will also organize an annual symposium that will be open to the public. In Specific Aim 2, the Administrative Core will be involved in setting up the external review of the Program Project Grant by appointing one external expert reviewer-consultant per year and one local non-collaboration expert. Finally, in Specific Aim 3, the Administrative Core aims to strengthen collaborations among the Core and Project Leaders in order to maximize the use of the various Cores by a maximum number of Projects. The Administrative Core is strategically positioned within the laboratory of Dr. Kelly, Program Director, which will enhance the efficiency and effectiveness of the tasks performed by the Administrative Core. All financial, budgetary, personnel, and payroll records will be coordinated by the Administrative Core in close collaboration with administrative personnel in Dr. Kelly's Laboratory and with grant administrative personnel at the Office of Sponsored Programs at The Scripps Research Institute. The Administrative Core will report on all those issues in regular Newsletters that will be sent to all Program Project Grant participants electronically and will ensure that the output of this Program Project Grant will be greater than the sum of the outputs of the separate Cores and Projects.
|
1 |
2013 — 2017 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Discovering Small Molecules Activators of Stress Responsive Signaling @ Scripps Research Institute
Program Director/Principal Investigator (Lasl, Fitst, MItldle): Morimoto, Richard I., PL Project 4-Kelly, Jeffery W. PROJECT SUMMARY (See inslrucUons): The maintenance of protein homeostasis, or proteostasis, involves balancing protein biosynthesis, folding, vesicular trafficking, degradation, etc., which we hypothesize is critical for healthy aging. Since the demands on subcellular compartments to maintain proteostasis change with aging and due to environmental stresses, stress-responsive signaling pathways have evolved to quickly adjust subcellular proteostasis network capacity to meet demand. Herein, we focus on the development and utilization of cell-based reporter screens to discover small molecules that can activate stress-responsive signaling pathways selectively. A dual luminescence reporter cell line is proposed to discover arm-selective unfolded protein response activators affecting proteostasis in the endoplasmic reticulum. A prior cell-based heat shock response (influencing cytosolic proteostasis) activator reporter screen has generated numerous small molecule leads and the same is expected from the arm-selective UPR activator screen. A plan is outlined to discern the stress responsive signaling pathway activation selectivity of these leads, to establish their therapeutic index, to identify the transcriptional and proteomic changes of select activators, and to identify and validate the target(s) of the highly ranked activators. We also propose to utilize small molecule-regulated destabilized domain-FOXO transcription factor fusions to deveiop a sensitive and selective cell-based reporter assay for eventual screening of small molecule activators of FOXO signaling. FOXO influences metabolic and proteostatic control. Selective stress-responsive signaling pathway activators will make it possible for the other investigators in this program project to learn which subcellular compartments are most important to maintain proteostasis in for healthy aging. Organelle-selective proteostasis enhancement in cell and animal models, coupled with the systems biology characterization of the proteostasis network as a function of aging and activator treatment should provide a clear answer regarding the influence of organelle-specific proteostasis on healthspan, the period of life where individuals are disease free-owing to the enhanced fitness of the proteome. RELEVANCE (See inslrucUons): The molecules produced in Project 4 and their utilization in testing the hypothesis that maintenance of the proteome over the lifespan of an organism is critical for healthspan represents a paradigm shift in aging research, which heretofore has largely focused on genome instability.
|
1 |
2015 — 2018 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interplay of Intrinsic and Extrinsic Effects of N-Glycans On Glycoproteostasis @ Scripps Research Institute
? DESCRIPTION (provided by applicant): About one-third of the proteome of eukaryotes traverses the cellular secretory pathway, and the majority of these proteins are N-glycosylated. Within the secretory pathway there is an elaborate network of chaperones, folding enzymes, and degradation machinery dedicated to maintaining glycoprotein homoeostasis, or glycoproteostasis. Failures of glycoproteostasis, either because of mutations in N-glycoproteins themselves or defects in the glycoproteostasis network, are responsible for many diseases, including cystic fibrosis. N-glycans affect glycoproteostasis through intrinsic mechanisms, by directly stabilizing glycoproteins and/or inhibiting their aggregation, and through extrinsic mechanisms, by mediating their interactions with the glycoproteostasis network. We have considerable experience studying the extrinsic role of N-glycans in glycoproteostasis maintenance through our studies of the folding and trafficking of glycoproteins associated with lysosomal storage diseases. We have also investigated in depth the intrinsic effects of N-glycans on protein folding and have carefully studied the effects of local sequence on the efficiency of protein N-glycosylation, and their influence on the N-glycan structures produced. In this proposal, we will fuse these areas of expertise to study how N- glycans intrinsically and extrinsically affect folding and trafficking vs. degradation decisions by the glycoproteostasis network. In Specific Aim 1, we will examine how the initial N-glycosylation event by oligosaccharyl transferase (OST) influences downstream trafficking vs. degradation (i.e., quality control) decisions by the glycoproteostasis network. We will explore the effect of N- glycosylation by OSTSTT3A vs. OSTSTT3B (where STT3A and STT3B are the two paralogs of the catalytic subunit of OST) on folding and trafficking vs. degradation decisions, by determining the effect of co-translational folding on substrate selectivity by OSTSTT3A vs OSTSTT3B, and by characterizing the interactomes of nascent glycoproteins and the various isoforms of OST itself. In Specific Aim 2, we will determine how the conformational properties of the N-glycoprotein determine the processing of N-glycans by glycoproteostasis network components. Many components of the glycoproteostasis network bind to N-glycoproteins in a bidentate fashion, interacting with both the N-glycan and the protein. This binding mode enables them to sense both the folding status of the protein and the mode and extent of N-glycan trimming, but it is unclear to what extent this sensing is a function of the immediate protein neighborhood of the N- glycan (neighborhood-local), the entire domain to which the N-glycan is attached (domain-local), or the domains that are remote from the N-glycosylation site (non-local).
|
1 |
2016 — 2020 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Probing the Biochemical Mechanisms of Amyloid Disease @ Scripps Research Institute
We seek to understand how the process of protein aggregation leads to the dysfunction and, ultimately, the death of post-mitotic tissue in the transthyretin (TTR) amyloid diseases. This understanding would enable the development of novel therapeutic strategies, the establishment of early diagnostic tactics, and the identification of biomarkers that quantify response to therapy. The TTR protein is secreted from the liver, it circulates in the blood, and its aggregation results in a primary neuropathy and/or cardiomyopathy, depending on the sequence(s) that misassembles. In Aim 1, we aspire to understand the structure-proteotoxicity relationship(s) driving the pathology of the TTR amyloidoses. We will isolate non-native TTR structures from patient plasma and subject them to structural characterization using atomic force microscopy and negative stain electron microscopy (EM). Non-native TTR structures that decrease upon tafamidis treatment (a disease-modifying kinetic stabilizer drug that stops TTR aggregation) and exhibit relevant cellular proteotoxicity will be further structurally characterized by cryo EM and by solid-state NMR. Cytotoxicity will be assessed in relevant primary cells and C. elegans, aiming to delineate the structures that are proteotoxic and preliminary mechanistic insights, while also assessing whether there are neurotoxic vs. cardiotoxic TTR structures. Moreover, we are developing novel peptide-based probes to quantify non-native TTR structures in blood to facilitate diagnosis and response to therapy across the >100 TTR sequences linked to pathology. In Aim 2, we will test the hypothesis that secretory pathway proteostasis network capacity in hepatocytes influences the folded structure, kinetic stability, and amyloidogenicity of secreted TTR. NMR evidence indicates the novel finding that an altered structural ensemble with enhanced TTR kinetic stability is afforded by folding TTR in a transcriptionally reprogrammed cellular proteostasis network. This aggregation resistance is due, in part, to the Hsp70 pathway, according to in vitro reconstitution experiments. We will continue to study the mechanism by which this and other proteostasis network pathways alter the structure, increase the kinetic stability, and reduce the aggregation propensity of secreted TTR in vivo. We will test the notion that wild type TTR produced by 10% of older males adopts a kinetically less stable, alternative tetramer structure that is more aggregation prone and thus leads to wild type TTR cardiomyopathy. We have developed a method to efficiently isolate TTR from healthy elderly vs. TTR amyloidosis patients to facilitate comparisons. That chaperone assisted folding can alter the folded structure of the client protein is a novel finding.
|
1 |
2018 — 2021 |
Kelly, Jeffery W Wiseman, Rockland Luke (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Discovering Small Molecule Activators of Stress-Responsive Signaling @ Scripps Research Institute
The maintenance of secreted protein homeostasis, or proteostasis, involves balancing protein biosynthesis, translocation across membranes, folding, degradation, etc., which we hypothesize is critical for healthy aging. Since the demands on secretory compartments to maintain proteostasis change with development, aging, and environmental stresses, mammals evolved the Unfolded Protein Response (UPR) stress-responsive signaling pathway, which transcriptionally adjusts secretory proteostasis network capacity to meet demand. Recent human genetic, chemical biologic, and in vivo evidence shows that activating the protective IRE1/XBP1s or ATF6 arms of the UPR has significant promise to ameliorate age-related declines in secretory proteostasis and correct imbalances associated with etiologically-diverse diseases, including systemic amyloid diseases, cardiovascular disorders, diabetes, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Few compounds exist to achieve arm-selective UPR activation, and those that do suffer from limitations that prevent their translational development. We have leveraged cell-based transcriptional reporter assays miniaturized for high-throughput screening (HTS), along with whole cell transcriptional and proteomic profiling to understand the selectivity of the transcriptional and translational response generated by our screening hits. We have elaborated promising compounds using medicinal chemistry to establish first-in- class small molecule `proteostasis regulators' that selectively activate the protective IRE1/XBP1s or ATF6 signaling arms of the UPR with improved potency and selectivity, and we seek their mechanism of action through multiple approaches. We will assess whether our proteostasis regulators can induce protective, arm- selective UPR activation in young and old animals. We have established collaborations to test the hypothesis that our IRE1/XBP1s and ATF6 activators will be useful for ameliorating pathologic imbalances in secretory proteostasis associated with multiple diseases, including the systemic amyloidoses, degenerative eye diseases, cardiovascular disease, and neurodegenerative disorders. Furthermore, we will show that these compounds pharmacologically ameliorate two pathologic phenotypes associated with Alzheimer's disease in cell culture models: i.e., the pathologic production of A? and A? oligomer-associated neuronal cytotoxicity. We will deliver to the scientific community the first well-characterized small molecules that preferentially activate the IRE1/XBP1s or the ATF6 UPR transcriptional programs with a defined potency and selectivity. These compounds have the potential to be widely employed as therapeutics for a spectrum of age-associated diseases. Importantly, these compounds will be made available to all scientists with disease models wherein pharmacologic IRE1/XBP1s or ATF6 activation has the potential to influence pathogenesis. The availability of these compounds offers the promise to broadly influence multiple aspects of scientific endeavor funded by the NIH, including basic science such as stem cell biology.
|
1 |
2019 — 2021 |
Kelly, Jeffery W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Proteostasis Regulator Pharmacology Core D @ Northwestern University
The overarching aim of Core D, in collaboration with Projects 1-4 and cores B and C, is to test the hypothesis that it is possible to partially reverse aging-dependent deficiencies in proteostasis network (PN) capacity (including those leading to pathology) by pharmacologic regulation of the PN employing small molecule proteostasis regulators. We will focus on heat shock response stress-responsive signaling pathway activators that regulate cytosolic PN capacity, unfolded protein response stress-responsive signaling activators that regulate secretory pathway PN capacity, and the antioxidant stress-responsive signaling pathway activators in Aim 1. Since stress-responsive signaling pathways generate an active transcription factor, we hypothesize that there will be a greater chance for an effective biological response from this evolved solution to correct proteostasis deficiencies, wherein all the components of interacting and competing PN pathways in a given subcellular compartment are up-regulated in the appropriate stoichiometry. These stress-responsive signaling pathways lead to powerful emergent functions that are only partially understood. In Aim 1, we will further develop a technology platform to validate the pharmacodynamics (PD; the study of what a drug does to the organism), selectivity, and mechanism of action of small molecule proteostasis regulators that function through activation of stress-responsive signaling pathways in multiple organisms. We will initially employ cell-based reporters of stress-responsive signaling pathway activation (with Core B), targeted RNAseq, followed by mass spectrometry-based proteomics (Core C activities), coupled to bioinformatics to validate the proteostasis regulators. We will also assess the pharmacokinetics (PK; the study of what the organism does to a drug (metabolism)) in multiple organisms, which will help us establish reasonable dosing regimens. PK will be assessed using liquid chromatography-mass spectrometry approaches. In Aim 2, we also seek to establish the utility of small molecule proteostasis regulators involved in enhancing the degradation of proteins, lipids and organelles, either through activation of autophagy or the ubiquitin proteasome system. We will generate PK and PD data for proteostasis regulators reported by others that activate the autophagy lysosomal pathway (degrades proteins, oligosaccharides, lipids and oligonucleotides) through an m-TOR independent mechanism and for proteostasis regulators to activate the ubiquitin proteasome system (degrades proteins). Collectively, the PK and PD data in human, murine and yeast cells and in C. elegans is important because: (1) it allows us to test and, therefore recommend, reasonable dosing regimens for proteostasis regulators, and (2) these data allow us to properly interpret experiments in model organisms, especially negative data. We will provide these validated proteostasis regulators to PIs of the projects, as well to labs working on complementary aging paradigms and aging-associated diseases to discern which proteostasis regulators correct aging-linked proteostasis deficiencies.
|
0.901 |
2020 |
Kelly, Jeffery W |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb's 'the Protein Folding in the Cell Conference' @ Federation of Amer Soc For Exper Biology
PROJECT SUMMARY This proposal seeks funding for US scientists to attend the FASEB Protein Folding in the Cell meeting to be held in Malahide, Ireland in July 2020. The focus of this meeting is on the basic science of defining protein folding pathways enabled by the proteostasis network, the functions of molecular chaperones and co- chaperones in folding versus degradation decisions, the latter mediated by the proteasome and lysosomal degradation. The program will also cover stress-responsive signaling pathways that sense the extent of proteome misfolding and aggregation and, if too high, initiate a transcriptional program to match proteostasis network capacity with demand in each subcellular environment. A decline in proteostasis capacity is especially prominent during aging and within several disease settings, including Alzheimer?s disease and related dementias of aging. Thus, the ability to manipulate proteostasis is highly relevant to enabling new therapeutic strategies for this important group of age-related neurodegenerative diseases. Other pathologies impacted by proteostasis imbalances include cancer, cardiovascular disease, metabolic syndrome and the lysosomal storage diseases. Studies in experimental model systems have revealed that small molecule proteostasis regulators, and combinations thereof, may be useful for ameliorating neurodegenerative diseases including Alzheimer?s disease and related dementias of aging?topics covered in the FASEB ?Protein Folding in the Cell? conference. The Protein Folding in the Cell conference focuses on the latest developments on how the native conformations of cellular and secreted proteins are achieved and maintained, and what happens in the cell (and multicellular organisms) when aberrant protein conformations arise, such as in Alzheimer?s disease and related dementias of aging. Special emphasis will be placed both on the formal presentations and on informal meetings for the transmission of information through discussion, group brainstorming sessions that catalyze the generation of new concepts and hypothesis generation, and the initiation of collaborations. An important goal of the meeting is to enable early career scientists to speak in the main oral presentation venue. Eighteen 10-min talks and at least five 20-min talks will be selected based on submitted abstracts and late breaking publications, being mindful of bringing under-represented scientists into the main program. Round table discussions over lunch will enable early career scientists to discuss topics like academic careers, industrial careers, grant writing, etc. with experts in the field. We will focus one working lunch with all attendees focused on the topic of attracting and retaining under-represented scientists into academia from the perspective of early career scientists. The purpose of this working lunch is to bring awareness to the challenges faced by female scientists and other minority scientists.
|
0.903 |
2021 |
Kelly, Jeffery W |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Pharmacologic Lysosomal Flux Activators to Ameliorate Alzheimer's Disease and Related Dementias @ Scripps Research Institute
A common molecular phenotype exists for most neurodegenerative diseases, including protein and/or protein- RNA aggregation, lipid level perturbations, mitochondrial dysfunction, lysosomal dysfunction, and neuro- inflammation. The literature provides evidence that this pathogenic signature of Alzheimer?s disease and related dementias can be normalized by genetic and pharmacologic activation of lysosomal flux?one mechanism to do this is called macroautophagy. To generate mechanistically diverse lysosomal flux activators, we screened 940,000 small molecules by a cell-based phenotypic screen to identify 108 validated small molecule hits that hastened lipid droplet clearance. Most known lysosomal flux activators function through inhibition of mTOR, which suppresses the immune system, putting the already vulnerable elderly population at higher risk for infectious disease. In this proposal we seek mTOR-independent lysosomal flux activators. In Aim 1 we employ traditional and novel assays to identify the targets of our hits, as well as their mechanisms of action. Whether these compounds induce macroautophagy (a cell component recycling pathway) or a specialized form of autophagy will be revealed by the proposed assays comprising Aim 1. We will also explore whether lysosomal flux activation occurs by other mechanisms, such as transcriptional reprogramming, or by a novel mechanism. The data generated in Aim 1 will guide prioritization of lysosomal flux activators that are best suited for ameliorating Alzheimer?s disease and related dementias. Aim 2 activities will scrutinize lysosomal flux activator dosing efficacy and dosing regimens in induced pluripotent stem cell-derived neurons, astrocytes and glial cells from hereditary Alzheimer?s disease patients and in brain organoids derived from these cells, as well as in brain cells and organoids lacking these mutations. Because autophagy recycles proteins, nucleic acids, oligosaccharides and lipids into their building blocks for reuse, it is a potential risk that enhancing lysosomal flux could degrade critical cellular components and therefore lead to on-mechanism toxicity. Organoids are well-suited for testing lysosomal flux activator multidosing regimens that avoid inducing cytotoxicity or cellular stress, while also normalizing the pathogenic Alzheimer?s disease-associated phenotypes present. We will use mass spectrometry-based proteomics and metabolomics / lipidomics to analyze the organoids after treatment by 20-30 prioritized lysosomal flux activators to learn how to dose so as to avoid cytotoxicity while normalizing the Alzheimer?s disease-relevant pathobiological phenotypes. Besides carrying out multiple biological replicates and appropriate statistical analyses, another way to ensure reproducibility and rigor is that we have distributed our lysosomal flux activators to multiple outside collaborators to carry out independent cellular assays and and efficacy assessments in models of Alzheimer?s disease. We hope to deliver a validated, mechanistically diverse, de-risked set of lysosomal flux activators as candidates to treat Alzheimer?s disease.
|
1 |
2021 |
Kelly, Jeffery W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Probing the Proteinopathy Component of Light Chain Amyloidosis Pharmacologically @ Scripps Research Institute
Project Summary Immunoglobulin light chain amyloidosis (AL) is a degenerative disease that putatively arises from light chain (LC) misfolding and aggregation. Full-length (FL) LCs are secreted from a clonally expanded plasma cell population in AL. Thus, AL patients suffer from both a plasma cell cancer and a LC misfolding and aggregation-associated proteinopathy that appears to compromise organ function, leading to progressive organ deterioration. Currently, AL is treated by repurposed multiple myeloma drugs that kill the clonal plasma cells secreting FL LCs. Mechanistically distinct therapeutic approaches are needed, particularly for patients with cardiac involvement who cannot tolerate the currently available chemotherapy regimens. In this proposal, we seek to produce lead candidates that ultimately would enable a clinical trial on a FL LC kinetic stabilizer targeting the proteinopathy component of AL. In Specific Aim 1, we hypothesize that the LC kinetic stabilizers to be synthesized in our hit-to-lead medicinal chemistry efforts can be dissected into four substructures?the ?anchor substructure?, the ?aromatic core?, the ?linker module? and the ?terminal aromatic component?. This hypothesis is based on 11 (FL LC)2?kinetic stabilizer crystal structures solved to date as well as the structure- activity relationship data resulting from the synthesis of over 300 FL LC2 kinetic stabilizers during the past 18 months. We will continue to use structure-based design principles that we learned developing the drug tafamidis, in addition to computational tools, especially the Schrödinger LiveDesign software, to replace the metabolically unstable and potentially toxic coumarin aromatic core and the diethyl aniline anchor substructure in our LC kinetic stabilizers, while introducing functionality to reduce albumin binding. The LiveDesign software utilizes a weighted combination of the predicted LogP values and docking scores to accurately predict albumin binding. In Specific Aim 2, we will develop FL LC2 plasma binding selectivity assays to validate our computational efforts to diminish albumin binding. First, we will add fluorescently labeled FL amyloidogenic LCs to pooled healthy donor plasma, along with proteinase K and a candidate kinetic stabilizer, and follow proteinase K endoproteolysis linked to FL LC2 conformational excursions chromatographically over time. If the kinetic stabilizer candidate selectively binds the amyloidogenic FL LC2 over all the other plasma proteins, including albumin, FL LC2 proteinase K endoproteolysis will be prevented?this assay is largely developed. Next, a subunit exchange assay will be developed to quantify pharmacologic FL LC2 kinetic stabilization in human plasma. This assay also quantifies kinetic stabilizer binding to albumin. A fluorescently labeled Cys214Ser FL LC2 variant facilitates subunit exchange experiments that afford the KD of kinetic stabilizer binding to the FL LC2 variants added to healthy plasma, as well as the kinetic stabilizer KD for binding to endogenous human albumin in plasma. These KDs will be used to generate binding selectivity ratios, i.e., KD of kinetic stabilizer binding to albumin / KD of kinetic stabilizer binding to the FL LC2, which we will maximize.
|
1 |