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Identification of neuronal membrane proteasome-derived peptides that modulate neuronal signaling
No full textThe novel neuroproteasome is localized to the neuronal plasma membrane where it degrades intracellular proteins into peptides that are released to the extracellular space. Selective inhibition of this neuronal membrane proteasome (NMP) complex ceased the release of peptides and rapidly attenuated neuronal transmission. Based on these findings, we hypothesize that these neuron-specific peptides mediate a novel form of communication through unique peptide-receptor interactions to promote intracellular signaling cascades relevant to neuronal development and function. Our work indicates that NMP peptides can rapidly induce N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx, leading to rapid and sustained phosphorylation of the well-defined activity-dependent transcription factor cAMP response element-binding protein (CREB). We also determined that the gene expression program drastically changes upon NMP peptide treatment of neurons with an increase in expression of immediate early genes (e.g., Fos, Npas4, Egr4) known to have critical neuroregulatory roles. These data support our current thinking that NMP peptides are endogenous and selective activators of synaptic NMDA receptors and are critical for promoting activity-dependent gene expression. This pathway is orthogonal to the classic neurotransmitters previously described to activate NMDARs and points to the NMP and its resulting peptides as potential key contributors to the proper function of the nervous system. However, the unique NMP-derived peptide sequences leading to neuronal activation are still poorly understood. Here, we show through an unbiased peptidomic approach that the NMP peptides have tremendous sequence diversity. The current ongoing effort is to identify unique active peptide sequences within this mixture that have distinct receptor specificity. Elucidating the mechanism of the NMP and its bioactive peptide products is crucial to understanding the role of this novel signaling process in the nervous system
Uncovering one-dimensional target search of ATP-dependent chromatin remodelers utilizing optical tweezers and single particle fluorescence microscopy
No full textATP-dependent chromatin remodelers actively control the locations and histone compositions of nucleosomes genome wide, regulating how and when the underlying genetic information is accessed. While all remodelers have at their cores similar DNA translocases, they exhibit different activities and nucleosome specificities due to differences in their motor domains and accessory subunits. This dissertation focuses on remodelers belonging to three of the four subfamilies: SWR1 (INO80 family), RSC (SWI/SNF family), and ISW2 (ISWI family) which are well characterized remodelers from Saccharomyces cerevisiae. These remodelers share specificity for nucleosomes at promoters, which are characteristically nucleosome depleted regions (NDRs). While a combination of factors contributes to the proper targeting of these remodelers to their substrate nucleosomes, one factor that remained to be investigated was the nature and importance of linear DNA-remodeler interactions. Here, I present biophysical evidence in favor of remodelers locating nucleosomes using facilitated diffusion, a process where DNA binding proteins find specific targets more efficiently by engaging in one-dimensional (1D) diffusion along DNA. The 1D diffusion of single, site-specifically labeled remodelers was imaged using confocal microscopy on DNA and nucleosome arrays stretched using optical tweezers. In Chapter 2, I characterize the 1D diffusion of SWR1, a remodeler that performs histone H2A.Z exchange, revealing that ATP-binding facilitates SWR1 target search in vitro by increasing its diffusivity on DNA. SWR1 diffusion was confined by nucleosomes, suggesting that the NDR may serve to corral 1D diffusion and target activity to flanking +1 and -1 nucleosomes. In Chapter 3, I examine the diffusive behaviors of RSC and ISW2, remodelers that regulate the accessibility of the transcription start site by sliding promoter nucleosomes. On naked DNA, these remodelers showed notable similarities and differences to SWR1. When diffusion was evaluated on sparsely dense nucleosome arrays, RSC and ISW2 both exhibit processive, unidirectional nucleosome sliding. Interestingly, the direction of approach via 1D diffusion biases the directionality of the resulting nucleosome sliding. Taken together, these studies support a model where 1D scanning on DNA may facilitate chromatin remodeler localization of NDR flanking nucleosomes and influence the directionality of resulting activity with implications in gene expression
Evaluating Improvements and Challenges in Affinity Chromatography for AAV Purification
Get PDFPurification process development is crucial to the Research and Development (R&D) sector in the biotechnology industry by designing and optimizing downstream unit operations capable of manufacturing safe treatments for clinical trials. Purification is crucial in manufacturing therapeutics such as monoclonal antibodies (mAbs), mRNA, viral vectors, and non-viral vectors. Recently, viral vector gene therapy modalities using adeno-associated viruses (AAVs) have been shown to be an effective therapeutic agent for both rare and common diseases in humans. One of the key polishing steps in the downstream manufacturing process of AAVs is affinity chromatography, where a ligand with specific binding affinity to an AAV serotype is coupled to a base matrix that separates AAVs from host cell contaminants produced in the cell culture process. Column resin is the most common affinity chromatography matrix, but it possesses mass transfer limitations. This report evaluated a prototype affinity membrane device coupled with AVB ligand designed to overcome these mass transfer limitations and decrease process times, while yielding comparable AAV recovery and purity to column resin.
The evaluations included dynamic binding capacity (DBC) studies, ligand lifetime studies, and buffer optimizations. The performance of the device was compared to control experiments using two common column resins with the same ligand as the device. The novel membrane device can bind at least 2e14 vg/mL_membrane and can undergo at least 3 cycles without a major binding capacity drop. The recovery of AAV is comparable to column resins; additional process development studies were required to increase recovery values because process parameters used for resin with the same ligand were not effective. A comparison of AAV product purity cannot be made because impurity profiles were not tested. Some recommendations for future work with the availability of more devices include more DBC studies, buffer optimization studies, and testing of the device performance with different AAV serotypes and load material. The prototype membrane device offers unique benefits over column resin for affinity chromatography in its fast flow rates, reusability, and potentially lower costs due to its “prepacked” design; however, the evaluations demonstrated the device does not provide significant enough process improvements that would favor its implementation in place of column resin for the tested material
Computational modeling, prediction, and design of Protein-Protein interactions
No full textProtein-protein interactions (PPIs) govern nearly all biological processes in human health and diseases, ranging from enzyme catalysis and inhibition, to signaling and gene regulation. Understanding the dynamics of protein interactions and the structure of protein complexes at an atomic level is key in delineating disease mechanisms, such as Huntington’s, Alzheimer’s, and cancer, and developing intervention strategies. Investigation of these structural complexes by experimental techniques is often expensive, laborious, and limited. Computational modeling provides an alternative route to elucidate structures and guide molecular engineering based on PPIs. A longstanding challenge limiting the accuracy of computational methods is the ability to predict binding-induced conformational changes during protein-protein association.
In my thesis, I address this challenge by creating new tools to predict atomistic models of flexible protein complexes. First, I develop a protein docking protocol that incorporates temperature replica exchange Monte Carlo (T-REMC) and backbone flexibility to mimic induced-fit approach of protein interactions. On a benchmark of 88 protein complexes with varying degrees of flexibility, this protocol, ReplicaDock 2.0, is the first method to successfully dock 62% of complexes with conformational changes up to 2.2 Å. Building on the success of ReplicaDock2.0, I extend it to develop a novel sampling approach, namely resolution exchange. In this approach, exchanges are performed between the full-atom and the centroid configurations to improve backbone sampling and escape entrapment in non-native minima. Finally, I conclude my docking methods development work by creating a pipeline that fuses AlphaFold (a deep-learning tool for protein sequence-to-structure prediction) with aforementioned docking techniques to develop a method for improved complex structure prediction.
In conjunction with development of foundational protein structure prediction tools, I equip docking tools to make contributions to human health and disease. First, to extend the functionality of MC approaches for capturing dynamics, I model the interactions between an outer membrane nutrient transporter (on bacterial cells) and a bacteriocin (Colicin B), and deduce the translocation pathway for Colicin B through the transporter. Next, I apply my knowledge of PPIs to create novel complex designs. I demonstrate a computational approach to create orthogonal interfaces with experimental validation for the PDGF signaling system. This technology has tremendous potential in regenerative medicine and therapeutic discovery as an orthogonal signaling system eliminates off-target risks (e.g., cancer) and promotes exclusivity.
In sum, my work has advanced our understanding and our ability to model and design flexible protein-protein interactions
Structural and functional conservation of TRP channels
No full textTransient Receptor Potential (TRP) ion channels act as cellular sensors that function in a variety of cell types to detect physical and chemical stimuli that may arise from either the environment or the cell itself. Mutations in TRP channels have been linked to heritable diseases, and they are attractive pharmacological targets for numerous other diseases.
Although solving the structures of membrane proteins has historically been challenging, many structures of TRP channels have been determined using cryo-electron microscopy (cryo-EM) since 2013 thanks to technical advances in detectors and in image processing. The plethora of structures includes different family members from different species under different conditions, a wealth of structural information for a family of channels that senses everything from temperature to acid to voltage to small molecules to ions. I leveraged the new availability of structural data to identify commonalities among the transmembrane (TM) domains of TRP channels and build a structure-based multiple sequence alignment. This structural comparison demonstrated that TRP channels share a common TM domain fold, with structural similarity being higher within subfamilies than between subfamilies. Examination of the pore domains revealed a conserved hydrophobic S6 gate constriction that appears to be too narrow to permit passage of hydrated cations in nearly all TRP channel structures solved to date. TRP channel selectivity filters share some general characteristics across the family, with unique features observed in monovalent-selective and divalent-selective filters that may contribute to their ion selectivity. Several hotspots for ligand binding in the TM domain were also identified and compared across the available structures.
Structural conservation in the cooling agent binding pocket indicated that TRPM4 might share sensitivity to the cooling agent icilin known to activate TRPM8. Patch clamp recordings in a heterologous expression system confirmed that TRPM4 activation by Ca2+ can be potentiated by icilin. A mutation equivalent to that known to increase icilin sensitivity in TRPM8 also increases icilin sensitivity in TRPM4, suggesting that the conserved icilin sensitivity is indeed mediated by the structurally-conserved ligand-binding pocket
THE RELATIONSHIP BETWEEN NIH PANDEMIC FUNDING AND RESEARCH PRODUCTIVITY
Get PDFThe COVID-19 pandemic has had a lasting effect on all aspects of society and required a quick response to stem the spread of the virus and mitigate its effects. In its efforts to combat the pandemic, the United States federal government appropriated research funding to the National Institutes of Health (NIH) to research the disease, its effects, and possible treatments. The NIH allocated this funding to its constituent institutes according to its mission, goals, and responsibilities in relation to the pandemic. This thesis utilized data from the NIH RePORTER website to analyze the overall changes in NIH funding for fiscal years 2017-2022. Additionally, an analysis of publication data for the same years was conducted to find a measure of research productivity over the course of the pandemic. The two analyses were then used to calculate the cost per publication for the NIH and its 27 constituent institutes. This study found that while the pandemic funding did result in a shift in NIH appropriations to its institutes, the publication costs were only indicative of a trend in research productivity for the NIH overall. A trend in publication costs could only be identified for specific institutes based on the relation of their focus of study to the pandemic
Dendrimer-Based Proteosomal Degradation of Indoleamine 2,3-Dioxygenase 1(IDO1)
No full textNeuroinflammation is an inflammation response in brain or spinal cord. It plays a key role in pathology of most central nervous system diseases such as Alzheimer’s, Stroke, Multiple Sclerosis, Autism, Traumatic Brain Injury, Gliomas. Therapeutics for neurological disorders take 35% longer to develop than drugs for other disorders since challenges remain on permeability across the blood-CNS barrier. PAMAM-OH is a highly water-soluble and eligible to pass through the blood-CNS barrier. Therefore, PAMAM dendrimer with hydroxyl termini(Generation 6) was employed as a platform to deliver 1-methyl-tryptophan which is the inhibitor of IDO 1. IDO 1 is an protein highly expressed in central nervous system when neuroinflammation occurs. Proteolysis Targeting Chimera strategy was applied to help better targeting and degrade the protein. The novel-designed conjugate drug was synthesized successfully and was tested in vitro. Through detecting the IDO 1 enzyme activity in two types of stimulated cell lines, the conjugates have had an enormous effect on inhibiting the enzyme. Therefore, the D-1MT PROTAC conjugates provide a practical option to make progress in dealing with Neuroinflammation
Chernoff Information in Community Detection
No full textIn network inference applications, it is desirable to detect community structure, i.e., cluster vertices into potential blocks. Beyond adjacency matrices, many real-world networks also involve vertex covariates that may carry information about underlying block structure. Since accurate inference on random networks depends on exploiting all available signal, we need scalable algorithms that can incorporate both network connectivity data and additional insight from vertex covariates. In addition, it can be prohibitively expensive to observe the entire graph in many real applications, especially for large graphs. Thus it becomes essential to identify vertices that have the most impact on block structure and only check whether there are edges between them given a limited budget.
To assess the effects of vertex covariates on block recovery, we consider two model-based spectral algorithms. The first algorithm uses only the adjacency matrix, and directly estimates the block assignments. The second algorithm incorporates both the adjacency matrix and the vertex covariates into the estimation of block assignments. We employ Chernoff information to analytically compare the algorithms’ performance and derive the information-theoretic Chernoff ratio for certain models of interest. Analytic results and simulations suggest that the second algorithm is often preferred: one can better estimate the induced block assignments by first estimating the effect of vertex covariates. In addition, real data experiments also indicate that the second algorithm has the advantage of revealing underlying block structure while considering observed vertex heterogeneity in real applications.
Moreover, we propose a dynamic network sampling scheme to optimize block recovery for stochastic blockmodel in the case where it is prohibitively expensive to observe the entire graph. Theoretically, we provide justification of our proposed Chernoff-optimal dynamic sampling scheme via the Chernoff information. Practically, we evaluate the performance of our method on several real datasets from different domains. Both theoretically and practically results suggest that our method can identify vertices that have the most impact on block structure so that one can only check whether there are edges between them to save significant resources but still recover the block structure
INVESTIGATING THE INFLUENCES OF INTRINSIC AND EXTRINSIC FACTORS ON FTSZ’S FUNCTION AND THEIR CONSEQUENCES FOR CELL DIVISION IN CAULOBACTER CRESCENTUS
No full textMost bacteria undergo cell division via active remodeling of the peptidoglycan (PG) cell wall, a mesh-like macromolecule that envelopes the cell and provides structure and protection. Remodeling is undertaken by PG synthases that are part of a multiprotein complex known as the “divisome.” Division requires precise regulation, which starts with the tubulin homolog FtsZ. Prior to division, FtsZ filaments assemble at mid-cell into a ring-like “Z-ring,” which serves as a marker for the site of a division and a scaffold to recruit the rest of the divisome. FtsZ then acts to distribute downstream factors about the division plane and regulate their activity to ensure proper constriction and cell separation. FtsZ exhibits GTPase dependent dynamics in the form of treadmilling along the inner circumference of the cell, and these dynamics are correlated with movement and activity of PG synthases. Using a combination of genetics and imaging, we have observed that in addition to affecting treadmilling, GTPase activity also guides proper Z-ring localization. Beyond its GTPase domain, FtsZ also contains a conserved C-terminal (CTC) peptide connected to the GTPase domain via a flexible and poorly conserved C-terminal linker (CTL). Our lab has previously established the CTL as essential for FtsZ’s regulation of PG synthase activity, evidenced by the formation of amorphous Z-rings and bulges in the cell envelope reminiscent of treatment with PG-targeting antibiotics upon expression of a variant lacking the CTL (ΔCTL). We leveraged this finding to investigate the contribution of other divisome components to this misregulation phenomenon and discovered that overabundance of the actin homolog FtsA, a membrane anchor for FtsZ, exacerbates the toxicity of this phenotype, implicating FtsA as a modulator in the FtsZ-to-PG synthase signaling pathway. Finally, we endeavored to observe the effects of combining these perturbations (ablating GTPase activity, removing CTL, and FtsA overabundance) to establish that each has distinct and specific influences on either Z-ring positioning or structure and function of the divisome. Collectively, this work has furthered our understanding of how FtsZ is regulated, both internally and by binding partners, and how these factors influence its ability to control the process of cell division
PARTICIPANT EXPERIENCES AND PERCEPTIONS OF BENEFITS AND RISKS IN VACCINE AND INPATIENT HEALTHY VOLUNTEER CLINICAL TRIALS AT THE CENTER FOR IMMUNIZATION RESEARCH
No full textBackground: Vaccine clinical trials are essential to ensure that immunizations are both safe and effective. Human volunteers provide the backbone of such trials, and sufficient numbers are required to produce the necessary data when evaluating a vaccine. Nevertheless, there is much debate and concern over the ethics of trials, especially those involving healthy volunteers, as well as significant hurdles in achieving target recruitment. There is a need for more information on how participants feel when participating in such trials and potential avenues to increase recruitment.
Methods: This dissertation utilized data from an interviewer-administered survey on participants of Phase 1 and 2 inpatient vaccine trials and human challenge studies (n=152), a self-administered electronic survey of participants in two Phase 2/3 COVID vaccine clinical trials (n=163), and in-depth interviews of guardians of pediatric COVID-19 vaccine trials (n=20) that all took place at the Johns Hopkins Center for Immunization Research in Baltimore, Maryland, USA. We analyzed descriptive statistics of the different studies and used Poisson regression with robust variance to examine risk factors for perception of any risk before and after participation.
Results: For most healthy volunteer clinical trial participants, the primary motivator to join was personal benefit, either compensation or access to a vaccine. Early-phase inpatient healthy volunteer trials were found to be generally composed of participants who identified as male, Black or African American, were of lower income, and had completed high school, whereas the outpatient COVID-19 studies were mostly female, White or Caucasian, had a masters or doctoral level degree, and were of higher income. Participants tended to believe the study was riskier before participating than after. The great majority of participants were glad they participated. Nevertheless, some participants still do not ask questions before joining a study.
Conclusions: Participants overwhelmingly reported being glad they joined the clinical trial they participated in, despite being different study phases and whether a vaccine was licensed or not. Considerations about personal benefits and improving understanding of clinical trials could be avenues to strengthen recruitment. Third party oversight of studies by IRBs continues to be imperative to ensure that studies are ethical and reasonable