2018 — 2019 |
Lander, Gabriel Cedric |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Conformational Ensembles Associated With Trpv3 Gating @ Scripps Research Institute
Transient receptor potential vanilloid (TRPV) channels are a family of nonselective cation channels that are expressed in skin, neuronal, and non-neuronal cells, and act as polymodal sensors for physicochemical changes in the environment, such as heat, endogenous or exogenous small molecules, and lipids. The thermoTRPV family is composed of four members (TRPV1-TRPV4), each displaying unique functional properties and modalities despite high sequence homology. The distinct features that govern these different modalities have been the subject of intense genetic, biophysical, and physiological studies. TRPV3, which is expressed at high levels in skin keratinocytes, plays vital roles in epidermal homeostasis, hair growth, and skin sensory functions. Precise regulation of this channel's activity is physiologically critical, as gain-of-function mutations (e.g. Gly573Ser) of the trpv3 gene in human and mice lead to itchy dermatitis and hairless phenotypes, while deletion of the gene leads to itch suppression and impaired skin growth and maintenance. TRPV3 inhibitors are currently being developed to relieve pruriceptive pruritus, inflammatory pain, and dermatological disorders, with one candidate in clinical trial. Biophysically, TRPV3 is distinctive amongst thermoTRPV channels in that TRPV3 sensitizes, rather than desensitizes, upon repeated application of stimuli. Despite the recent progress in structural studies of TRP channels, the structure of TRPV3 is unknown, and thus our understanding of the unusual molecular mechanisms of TRPV3 gating has been limited. Importantly, the unique sensitization properties of TRPV3 provide a unique opportunity to observe the open, sensitized channel conformation, which is not feasible using other thermoTRPs. The goal of the proposed research is to describe, at an atomic level, how the human TRPV3 channel responds to chemical stimuli, and how structural rearrangements are coordinated with channel opening. Our preliminary data suggest that conformational rearrangements within the ring of cytoplasmic ankyrin repeat domains (ARDs) are directly linked to TRPV3 channel sensitization and activation. Using a combination of cryo-electron microscopy, mutagenesis, and electrophysiology, we will test this hypothesis in TRPV3 and probe its mechanistic similarity and difference across the thermoTRPV family of ion channels through comparisons with TRPV1 and TRPV2, which desensitize upon repeated stimuli. Successful completion of our aims will provide atomistic descriptions of TRPV3 channel gating, serving as a platform for the development of future drugs targeting human TRPV3. Furthermore, our results will contribute to a general understanding of the gating mechanisms of thermoTRPV channels; importantly explaining how the ARDs mechanistically influence thermoTRPV channel opening, a question that remains a mystery in the field.
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2019 — 2020 |
Lander, Gabriel Cedric |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Defining the Molecular Mechanisms Underlying Mitochondrial Proteostasis @ Scripps Research Institute
Project Summary A critical factor in defining mitochondrial dysfunction during normal aging and age-associated disease is the maintenance of mitochondrial protein integrity (also referred to as protein homeostasis or proteostasis). Mitochondrial proteostasis is primarily regulated by ATP-dependent quality control proteases, including the soluble proteases, LON and CLPXP, and the membrane associated proteases, YME1L and AFG3L2. These quality control proteases regulate all aspects of mitochondrial function including energy metabolism, lipid synthesis, and apoptotic signaling. Mitochondrial quality control proteases adopt a similar ?stacked ring? organization, wherein evolutionarily similar AAA+ ATPase domains undergo ATP-dependent rearrangements to translocate protein substrates through a central pore to a proteolytic chamber for cleavage. Despite the evident structural and mechanistic similarities, mitochondrial proteases exhibit distinct proteolytic activities that allow them to regulate specific mitochondrial function. A consequence of the distinct functions of these proteases is that genetic or age-related alterations in the activity of specific proteases distinctly influences mitochondrial and cellular physiology in the context of organismal aging. Thus, an important question is ?How do these mitochondrial quality control proteases use a similar architecture to differentially influence specific aspects of mitochondrial biology?? Here, we hypothesize that unique structural features in each of these proteases have been incorporated into a generally conserved mechanism of ATP-dependent protease activity, endowing mitochondrial quality control proteases the capacity for unique biologic function. We solved the first near atomic resolution cryo-electron microscopy (cryo-EM) structure of the catalytic core of the yeast YME1L homolog YME1 bound to nucleotides and a peptide substrate, revealing the molecular mechanism responsible for ATP-dependent substrate engagement and translocation into the proteolytic chamber. We are now defining similar substrate-bound structures for the soluble AAA+ proteases LON and CLPXP using an analogous cryo-EM approach. Furthermore, we are establishing a novel unnatural amino acid platform to isolate the full-length membrane integrated (IM) proteases YME1L and AFG3L2 for structure determination by cryo-EM. By comparing structures of these evolutionarily related proteases, we are identifying the shared mechanisms that drive substrate translocation, as well as the unique structural features critical for their specific protease activities. These differences will reveal the molecular mechanisms by which aging or mutation may influence the activity of proteases, and identify new opportunities to therapeutically influence mitochondrial proteolytic activity to prevent aging- or disease-associated mitochondrial dysfunction by targeting specific aspects of protease structure.
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2021 |
Lander, Gabriel C |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of a Pipeline For Parallel Elucidation of Protein Structures @ Scripps Research Institute
Advances in biophysical technologies have accelerated our ability to probe the mechanisms of even the most complex cellular systems, and such studies have enabled researchers to design modifications to known protein structures and design completely new proteins. This ?protein design? technology has given rise to an ability to manipulate protein structures as a means of improving on or introducing new medical diagnostics and therapeutics. The bases of these studies rely on computational modeling of protein candidates, although the accuracy of protein structure prediction, protein de novo design, and single-mutation effects prediction remain below the threshold for many use cases, such as structure-guided drug design and rational enzyme engineering. Thus, success of a protein engineering effort relies on high-resolution structure determination, which involves laborious screening and optimization in order to obtain stable proteins or active enzyme variants. However, our ability to observe protein structure using common structure determination strategies (X-ray crystallography, NMR, and cryo-electron microscopy (cryo-EM)) lags far behind our ability to design and produce new sequences, creating a knowledge gap that prevents biochemists from accessing the range of protein functions seen in nature. While current technologies enable rapid synthesis of hundreds of proteins with varied sequences, there do not exist technologies for rapid structural characterization of these generated proteins. The ability to obtain high- resolution structural information for hundreds of sequences in parallel would provide invaluable insights in protein engineering methods. Importantly, rapid structure determination would enable structural characterization of genetic variation in the human genome underlying disease by enabling the structural and mechanistic interpretation of rare and de novo disease-related variants. Cryo-EM enables numerous high-resolution structures to be determined from a small amount of sample without requiring homogeneity, an aspect of this method that we plan to exploit for parallel elucidation of protein structures. We will establish the feasibility of this technique for rapidly investigate the structures of engineered protein libraries, where the molecular weight range is near or below the lower detection limit of cryo-EM. We will also probe the limits of our ability to identify the location and structural impact of tested mutations at limited structural locations, such as active sites. We will explore the feasibility of our parallel structure determination approach in two aims: Aim 1 will identify the limit of current single-particle analysis methods to discriminate between structurally similar protein complexes. Aim 2 will implement machine learning algorithms to push the current limits of classification using a combination of synthetic and real data. These exploratory studies will pave the way to rapid structure determination of multiple protein complexes from a single cryo-EM experiment, providing the ability to rapidly obtain high-resolution structures for many engineered proteins, thereby enabling unprecedented design and testing feedback cycles to help treat human disease.
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2021 |
Lander, Gabriel C |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Extending the Limits of Cryo-Em to Better Understand Ttr Misfolding and Aggregation @ Scripps Research Institute
Appl ID: 9981223 Grant Number : 1R21 AG067594-01 PI Name : Lander, Gabriel Grant Title : Extending the limits of cryo-EM to better understand TTR misfolding and aggregation PROJECT SUMMARY The conversion of natively folded proteins into non-functional aggregates is associated with a wide range of age-related degenerative diseases, including Alzheimer?s Disease (AD). Of great concern is the prediction that these diseases will become more prevalent in prevalence as our nation?s life expectancy increases. The development of therapeutics to prevent or reverse the protein misfolding events implicated in degenerative diseases has thus become the focus of many investigative efforts. The protein transthyretin (TTR), a thyroxine and holoretinol carrier protein exported to cerebrospinal fluid (CSF) and serum, is one such protein that demonstrates increased propensity to adopt a non-native fold and form insoluble aggregates with age. This process can occur in the native, wild type form of the TTR protein, and is responsible for wild type TTR amyloidosis (also known as senile systemic amyloidosis), which causes restrictive cardiomyopathy. Notably, there is evidence that TTR interacts with the A? peptide, thereby preventing A? fibril formation and aggregation. Studies demonstrating that the CSF of AD patients contain substantially lower concentrations of TTR than in the CSF of age-matched non-AD individuals supports a neuroprotective role of TTR. We posit that age-related TTR misfolding and aggregation abolishes the capacity of TTR to prevent A? fibril formation and the subsequent onset of AD. In order to better understand how a natively folded wild type TTR protein becomes predisposed to misfolding events, we seek funding to develop structural approaches to study both the native and aberrant forms of TTR. Detailed structural information regarding the destabilization and non-native oligomerization of this protein will profoundly impact our understanding of TTR misfolding and fibrillogenesis, and could lead to the development of more potent TTR stabilizing drugs that could restore neuroprotective properties of TTR in AD patients. The first aim we will push the limits of size and resolution attainable by cryo-electron microscopy to examine the high-resolution three-dimensional structures of destabilized TTR tetramers and compare them to tetramers that are stabilized by small molecule ligands, revealing how the incorporation of misfolded subunits or the improper incorporation of natively folded subunits impact TTR stability at an atomic level. The second aim will define the architecture of TTR aggregates with atomic precision in order to shed light on the assembly pathways and how different oligomeric states differentially contribute to a variety of distinct pathogeneses. Notably, since perturbations of the native folding pathway of wild type proteins are the likely cytotoxic drivers of AD, the findings of this work will likely have far-reaching impact beyond TTR amyloidoses.
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2021 |
Lander, Gabriel C Wiseman, Rockland Luke [⬀] |
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. |
Impacting Mitochondrial Function Through Altered Protease Activity @ Scripps Research Institute
SUMMARY Mitochondrial dysfunction is a pathologic hallmark in the onset and pathogenesis of nearly all neurodegenerative diseases. One of the primary determinants in dictating mitochondrial function is the activity of inner membrane (IM) proteases including the ATP-dependent AAA+ zinc metalloproteases YME1L and AFG3L2 and the ATP-independent zinc metalloprotease OMA1. These proteases regulate many different aspects of mitochondrial biology and function to protect mitochondria from pathologic insults. However, imbalances in the activity of IM proteases induced by genetic or environmental factors are implicated in the pathogenesis of etiologically-diverse diseases including many neurodegenerative disorders. Despite this, the molecular mechanisms by which IM proteases regulate mitochondrial biology remain poorly understood. Here, we are applying a structure-driven approach to determine the molecular mechanisms by which IM proteases regulate mitochondria in the context of health and disease. We previously solved the first high-resolution structures of the IM AAA+ proteases YME1 and AFG3L2. Our structures showed that these two proteases employ a conserved nucleotide-driven, hand-over-hand mechanism to translocate substrates into a privileged proteolytic chamber for proteolysis. Surprisingly, we also identified unique structural features of YME1 and AFG3L2 that integrate into this conserved translocation mechanism to distinctly influence protease activity and stability. Here, we hypothesize that these unique structural differences endow IM proteases with different mechanistic and biologic functions important for their regulation of mitochondria. To address this, we are using a combination of cryo-electron microscopy and cell biology to determine how structural differences in IM AAA+ proteases influence their mechanochemical cycle and enable proteases to perform distinct biological functions. This will reveal new insights into the molecular mechanisms by which IM AAA+ proteases regulate mitochondria in health and disease. Furthermore, we are extending this study utilizing both functional genomic and structural approaches to establish a structure-function relationship that explains the activation and proteolytic activity of the ATP-independent, stress-activated IM protease OMA1 ? a protease whose dysregulation is implicated in the pathologic mitochondrial dysfunction associated with many human diseases. Through these efforts, we will define how IM proteases utilize distinct structural features to perform the myriad of biological functions required for the proper regulation of mitochondrial proteostasis and function. Furthermore, we will reveal new insights into the pathologic and potentially therapeutic implications of altered mitochondrial IM protease activity in human disease and identify new opportunities to pharmacologically target IM proteases to mitigate mitochondrial dysfunction associated with many neurodegenerative disorders.
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