Browsing by Author "Kusmierczyk, Andrew"

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    Alternative Assembly Pathways of the 20S Proteasome and Non-canonical Complexes
    (2018-12) Panfair, Dilrajkaur; Balakrishnan, Lata; Kusmierczyk, Andrew; Randall, Stephen; Rubenstein, Eric; Anderson, Gregory
    The 20S proteasome, a multi-subunit protease complex, present in all domains of life and some orders of bacteria, is involved in degradation of the majority of cellular proteins. Structurally, it is made of α and β subunits arranged in four heptameric rings, with inner two β-rings sandwiched between outer two α-rings. The 20S proteasome in prokaryotes usually has one type of α and one type of β subunits, whereas eukaryotes have seven distinct types of α and seven distinct types of β subunits. Unlike the highly conserved structure of proteasome, its assembly pathway is different across the domains. In archaea and eukaryotes, proteasome assembly begins with α subunit interactions leading to the α-ring formation. By contrast, bacterial proteasome assembly pathway bypasses the α-ring formation step by initiating assembly through an α and β subunit interaction first. These early interactions are not well understood due to their highly rapid and dynamic nature. This dissertation focused on understanding the early events in proteasome assembly and contributed three significant findings. First, the archaeal proteasome assembly can also begin without formation of α-rings, demonstrating the coexistence of a bacterial-like assembly pathway. Second, a novel assembly intermediate was identified in yeast, and its composition argues for the presence of a similar α-ring independent assembly pathway. Third, the assembly chaperone Pba3-Pba4 prevents the formation of high molecular weight complexes arising from spontaneous and non-productive interactions among the α subunits. These findings provide a broader understanding of proteasome biogenesis and suggest considering proteasome assembly event as a network of interactions rather than a linear pathway. The results also shed light on assembly chaperone’s contribution in increasing the efficiency of proteasome assembly by streamlining the productive interactions.
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    Cascades of genetic instability resulting from compromised break-induced replication
    (2013) Vasan, Soumini; Malkova, Anna; Atkinson, Simon; Kusmierczyk, Andrew
    Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.
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    Functional dissection of ERD14 phosphorylation-dependent calcium binding activity
    (2014-12-11) Chacha, Allen R.; Randall, Stephen Karl, 1953-; Watson, John; Kusmierczyk, Andrew
    Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.
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    Identification of the Pba1 and Pba2 Binding Sites on 20S Core Particle Intermediates
    (2013-07-12) Hammack, Lindsay Jo; Kusmierczyk, Andrew; Malkova, Anna; Randall, Stephen Karl, 1953-; Atkinson, Simon
    The proteasome is responsible for breaking down the majority of the proteins in the cell. However, a complete understanding of how this large multi-subunit protease is assembled is currently lacking. Proper and timely assembly of the proteasome is critical for the functioning of the ubiquitin-proteasome pathway, defects in which have been associated with several different cancers. A recently discovered heterodimeric proteasome assembly chaperone, Pba1p-Pba2p, has been suggested to prevent the assembly process from straying off path. Pba1p-Pba2p associates with proteasomal assembly intermediates via C-terminal HbYX motifs. The HbYX motif is a tri-peptide sequence containing a hydrophobic residue (Hb) followed by a tyrosine (Y), then any amino acid (X). This motif was originally identified in proteasomal activators, and shown to mediate the association of activators with the proteasome by inserting into intersubunit pockets on either end of the proteasome. There are seven unique intersubunit binding pockets, located between neighboring α subunits on the proteasome, to which a HbYX-containing protein can bind; which of these pockets Pba1p-Pba2p binds to remains elusive. I attempted to identify where Pba1p and Pba2p bind via a crosslinking approach. Specific residues were mutagenized to cysteines on Pba1p, Pba2p, and the individual α subunits in order to generate crosslinkable species. By exposing yeast cells expressing these crosslinkable proteins to mild oxidizing conditions, I attempted to trap the Pba1p and Pba2p α intersubunit pocket interactions. In order to optimize crosslinking conditions, the assay was modified several ways. Additionally, measures were taken to increase detection of the crosslinked species via immunoblotting. Despite the efforts to improve the crosslinking and detection, I was unable to successfully detect a crosslinked species. However, crosslinking is a reasonable method to identify the Pba1p and Pba2p proteasomal binding sites, having been successfully used to identify binding sites for other HbYX-motif-containing proteins; further assay optimization should yield Pba1p and Pba2p proteasomal crosslinks.
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    Investigating the early events in proteasome assembly
    (2014) Ramamurthy, Aishwarya; Kusmierczyk, Andrew; Atkinson, Simon; Randall, Stephen Karl, 1953-
    Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.
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    Investigation of the potential bacterial proteasome homologue Anbu
    (2014-09-08) Suknaic, Stephen R.; Kusmierczyk, Andrew; Randall, Stephen Karl, 1953-; Anderson, Gregory G.
    Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.
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    Molecular and Cellular Mechanisms Leading to Similar Phenotypes in Down and Fetal Alcohol Syndromes
    (2013-08-22) Solzak, Jeffrey Peter; Roper, Randall J.; Marrs, James; Kusmierczyk, Andrew; Atkinson, Simon
    Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from cognitive impairment to craniofacial abnormalities. While DS originates from the trisomy of human chromosome 21 and FAS from prenatal alcohol consumption, many of the defining characteristics for these two disorders are stunningly similar. A survey of the literature revealed over 20 similar craniofacial and structural deficits in both human and mouse models of DS and FAS. We hypothesized that the similar phenotypes observed are caused by disruptions in common molecular or cellular pathways during development. To test our hypothesis, we examined morphometric, genetic, and cellular phenotypes during development of our DS and FAS mouse models at embryonic days 9.5-10.5. Our preliminary evidence indicates that during early development, dysregulation of Dyrk1a and Rcan1, cardinal genes affecting craniofacial and neurological precursors of DS, are also dysregulated in embryonic FAS models. Furthermore, Caspase 3 was also found to have similar expression in DS and FAS craniofacial neural crest derived tissues such as the first branchial arch (BA1) and regions of the brain. This may explain a developmental deficit by means of apoptosis. We have also investigated the expression of pAkt, a protein shown to be affected in FAS models, in cells located within the craniofacial precursor of Ts65Dn. Recent research shows that Ttc3, a gene that is triplicated and shown to be overexpressed in the BA1 and neural tube of Ts65Dn, targets pAkt in the nucleus affecting important transcription factors regulating cell cycle and cell survival. While Akt has been shown to play a role in neuronal development, we hypothesize that it also affects similar cellular properties in craniofacial precursors during development. By comparing common genotypes and phenotypes of DS and FAS we may provide common mechanisms to target for potential treatments of both disorders. One of the least understood phenotypes of DS is their deficient immune system. Many individuals with DS have varying serious illnesses ranging from coeliac disease to respiratory infections that are a direct result of this immunodeficiency. Proteasomes are an integral part of a competent and efficient immune system. It has been observed that mice lacking immunoproteasomes present deficiencies in providing MHC class I peptides, proteins essential in identifying infections. A gene, Psmg1 (Dscr2), triplicated in both humans and in Ts65Dn mice, is known to act as a proteasome assembly chaperone for the 20S proteasome. We hypothesized that a dysregulation in this gene promotes a proteasome assembly aberration, impacting the efficiency of the DS immune system. To test this hypothesis we performed western blot analysis on specific precursor and processed β-subunits of the 20S proteasome in thymic tissue of adult Ts65Dn. While the β-subunits tested displayed no significant differences between trisomic and euploid mice we have provided further insight to the origins of immunodeficiency in DS.