Research

 

Molecular Mechanism of Stress Signaling Pathways

 

We are currently focused on understanding the UPR at the ER and translational remodeling in the cytoplasm; two pathways that respond to different cellular stressors and help restore cellular homeostasis. Even with decades of research on these systems, there is a knowledge gap in the molecular details of these pathways. There are no high-resolution structures of the proteins and protein complexes involved in these pathways with only snapshots of individual components available. We employ cryoEM and cryoET approaches to understand the stress response pathways at the molecular, organellar and cellular levels.

 

Stress Signaling Maladaptation in Disease

 

Defects in stress signaling pathways are linked to a dozen different diseases including cancer, metabolic disorders and neurodegeneration. For example, cancerous cells have high protein folding and metabolic loads and the protein quality control sensors are hyperactive to the benefit of these cells. Additionally, different disease conditions elicit different stresses such as hypoxia, protein misfolding and lipid imbalance, and it is not known which pathways are critical in which stress condition. We are focused on understanding the mechanism of protein quality control hyperactivation in disease.
Rewiring Stress Signaling Pathways with Targeted Drug Design

 

Given the critical role of stress signaling in disease conditions, many therapeutic strategies have targeted these pathways. However, the lack of molecular information has led to limited success thus far. Our lab seeks to overcome this challenge by leveraging the molecular information combining rational design with Rosetta-based computational modeling and machine learning. Given that most drugs suffer from off-target effects, our structure-guided approach offers specificity and efficacy in targeting protein quality control pathways. Moreover, our experience in developing structure-guided therapeutics (see past projects) gives us the technical expertise to tackle this problem.

 

 

Past Research Projects:

 

Design of an Aerosolized Spray against SARS-CoV2 Virus

 

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). We have developed nanobodies that disrupt the interaction between Spike and ACE2 by screening a yeast surface-displayed library of synthetic nanobody sequences. We then used cryoEM and X-ray crystallography to obtain the atomic level details of the nanobody. One nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. We utilized biochemical assays such as affinity maturation and structure-guided design to develop an ultra-stable aerosolized nanobody capable of preventing SARS-CoV2 infections.

 
 

 

 

Structure-guided Inhibitors of alpha-Synuclein
Aggregation in Synucleinopathies

 

a-Syn is a protein that forms amyloid fibrils in Parkinson’s disease and several related diseases together known as Synucleinopathies and, it is known that pathology progresses through interconnected brain regions a phenomenon known as seeding where preformed small fibrils can template the formation of fibrils. A short segment (residues 68-78) is known to be necessary for full-length α-syn aggregation both in vitro and in vivo. As a collaborative effort, we determined the atomic structure of a short segment known to be necessary for full-length α-syn aggregation both in vitro and in vivo (Rodriguez et al. Nature 2015). We then utilized the structure to design a line of peptide inhibitors that prevent α-syn aggregation and seeding (Sangwan et al. eLife 2020). Our approach displayed the potential of rational structure-based drug design to halt protein aggregation.

 

 

Molecular Mechanism of SOD1-linked Cytotoxicity in ALS

 

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. As a graduate student, Smriti determined the structure of a short segment of SOD1 by X-ray crystallography. The segment adopts a corkscrew-like fold forming oligomers. Using mutagensis approaches, she confirmed that the oligomeric conformation was crticial for SOD1 cytotoxicity in primary motor neurons and in a zebrafish model of ALS (Sangwan et al. PNAS). She also determined 3 crystal structures of overlapping segments from SOD1. The crystal structures reveal three different architectures: corkscrew oligomeric structure, non-twisting curved sheet structure and a steric zipper proto‐filament structure (Sangwan et al. Protein Science). Her work highlights the polymorphism of the segment 28–38 of SOD1 and identified the molecular features of amyloidogenic entities.