Browsing by Subject "Ubiquitin"

Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age.

Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear.

Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.

Parkinson’s disease (PD), a common neurodegenerative disorder, can result from defective proteostasis mechanisms that induce neuronal toxicity. A common pathological hallmark of PD is the disruption of protein transport and trafficking between organelles within the neuron. To date, there has been some success in identifying small drug-like molecules that can restore defective trafficking pathways. These compounds are often identified through phenotypic screening in cellular models of PD toxicity, but, for many, the specific target of the compound and its mechanism of action are not fully understood. One such compound, identified in a screening effort in the laboratory of Susan Lindquist, was recently shown to alleviate multiple phenotypic markers of PD toxicity. In particular, the compound was shown to alleviate markers of toxicity induced by aggregation of α-synuclein, a protein that is genetically linked to PD. The activity of this compound, which contains a characteristic N-arylbenzdiimidazole (NAB) scaffold, was shown to depend upon E3 ubiquitin ligase Nedd4. Nedd4 has been previously implicated in PD toxicity as it regulates α-synuclein aggregation and proteostasis through ubiquitin signaling, but the mechanism of NAB compounds as modulators of Nedd4 was not elucidated. To this end, a series of biochemical and biophysical analyses were employed to understand the binding and mechanism of NAB2, the most potent NAB derivative, as a ligand of Nedd4. These experiments revealed that NAB2 binds to Nedd4 with high apparent affinity in the nanomolar range but does not induce changes in Nedd4 enzymatic activity in vitro. As Nedd4 activity is dependent upon protein-protein interaction and is tightly regulated at a cellular level, a proteomics-based evaluation of ubiquitination was pursued to study the effect of NAB2 treatment on the global ubiquitylome. This analysis revealed that induction of α-synuclein toxicity dramatically remodels the ubiquitylome, and NAB2 treatment induces small but phenotypically relevant changes in protein ubiquitination. Through this effort, a previously unrecognized Nedd4 substrate, trafficking scaffold protein TFG, was identified to be ubiquitinated in a NAB-dependent manner.

To further expand our understanding of the NAB2 mechanism in alleviation of α-synuclein toxicity, an unbiased chemoproteomic approach was employed to identify NAB2 targets across the proteome. This effort revealed small GTPase Rab1a, a regulator of endoplasmic reticulum (ER)-to-Golgi body transport, as an additional putative target of the NAB scaffold. This result is particularly promising as ER-to-Golgi transport is disrupted in PD toxicity and restored by NAB2 treatment. Further analysis of NAB2/Rab1a binding indicate that NAB2 binding occurs in a nucleotide-dependent manner, and NAB2 treatment phenocopies Rab1a overexpression by improving the viability of α-synuclein toxic cells. While the functional link between Nedd4 and Rab1a is not yet clear, the efforts toward understanding the NAB mechanism of action have revealed a protein network involved in NAB2-dependent rescue of trafficking defects and PD toxicity. Cumulatively, these results expand our understanding, at a molecular level, of PD toxicity and small molecule rescue thereof, enabling future efforts to target these proteins for development or optimization of neuroprotective compounds.

Intracellular bacteria such as Chlamydia inhabit a single-membrane vacuolar compartment termed the “inclusion”. Chlamydia inclusions offer an escape from host immune defenses and a dedicated spaces for replication. Viable inclusion membranes must be modified by Chlamydia to sequester nutrients and camouflage themselves from host immune defenses that otherwise sense, contain, or eradicate microbial invaders. C. trachomatis, a human-adapted pathogen responsible for widespread sexually transmitted disease, forms inclusions that are protected from gamma-interferon (IFNγ)-induced cell-autonomous immunity. However, it is largely unknown how the unique architecture of host and bacterial components that localize to inclusion membranes impact chlamydial pathogenesis during IFNγ-stimulated conditions. This dissertation details a novel C. trachomatis secreted effector, the gamma resistance determinant or “GarD”, that shields inclusions from cell-autonomous immunity. Specifically, GarD forms a perimeter on inclusion membranes to prevent attack by the IFNγ-inducible enzyme RNF213, which labels GarD-less inclusions with linear poly-ubiquitin protein chains as a signal for elimination. Therefore, GarD operates as an essential antagonist of RNF213-mediated ubiquitylation and allows C. trachomatis to endure human cell-autonomous defenses. Future and ongoing work considers the mechanics of how GarD, RNF213 and additional bacterial and host cell factors affect pathogenesis of Chlamydia species in mammals. Overall, the novel GarD-RNF213 axis is an important expansion of our knowledge of chlamydial pathogenesis and immune responses to vacuole-residing pathogens.

A molecular depiction of the assembly, interaction and regulation of protein complexes is essential to the understanding of biological functions of protein complexes. Structural analysis of protein complexes by Nuclear Magnetic Resonance (NMR) has relied heavily on the detection and assignment of intermolecular Nuclear Overhauser Effects (NOEs) that define the interactions of protons at the molecular interface. Intermolecular NOEs have traditionally been detected from 3-D half-filtered NOE experiments by suppressing intramolecular NOEs prior to NOE transfer. However, due to insufficient suppression of undesirable signals and a lack of dispersion in the H dimension, data analysis is complicated by the interference of residual intramolecular NOEs and assignment ambiguity, both of which can lead to distorted or even erroneously packed protein complex structures. Leveraging the recent development of fast NMR technology based on sparse sampling in our lab, we developed a strategy for reliable identification and assignment of intermolecular NOEs using high resolution 4-D NOE difference spectroscopy. Spectral subtraction of individually labeled components from a uniformly labeled protein complex yields an "omit" spectrum containing only intermolecular NOEs with little signal degeneracy.

The benefit of such a strategy is first demonstrated in structural analysis of a homooligomeric protein complexes, the foldon trimer. We show that intermolecular NOEs collected from the 4-D omit NOE spectrum can be directly utilized for automated structural analysis of the foldon trimer by CYANA, whereas intermolecular NOEs derived from 3-D half-filtered NOE experiments failed to generate a converged structure under the same condition.

Such a strategy was further demonstrated on a heterodimeric protein complex in translesion sysnthesis (TLS), a DNA damage tolerance pathway. The TLS machinery consists of several translesion DNA polymerases that are recruited to the stalled replication fork in response to monoubiquitinated proliferating cell nuclear antigen (PCNA) in order to bypass DNA lesions encountered during genomic replication. The recruitment and assembly of translesion machinery is heavily dependent on ubiquitin-binding domains, including ubiquitin-binding motifs (UBMs) and ubiquitin-binding zinc fingers (UBZs) that are found in translesion DNA polymerases. Two conserved ubiquitin-binding motifs (UBM1 and UBM2) are found in the Y-family polymerase (Pol) &iota, both of which contribute to ubiquitin-mediated accumulation of Pol &iota during TLS. Although the Pol&iota UBM2-ubiquitin complex has been previous reported by our lab and others, the Pol &iota UBM1-ubiquitin complex has remained a challenge due to significant signal overlap in conventional 3-D NOE spectroscopy. In order to determine the molecular basis for ubiquitin recognition of Pol &iota, we solved the structures of human Pol &iota UBM1 and its complex with ubiquitin by 4-D fast NMR, revealing a signature helix-turn-helix motif that recognizes ubiquitin through an unconventional surface centered at L8 of ubiquitin. Importantly, the use of 4-D omit NOE spectroscopy unambiguously revealed an augmented ubiquitin binding interface that encompasses the C-terminal tail of UBM1.

4-D omit NOE spectroscopy was also used to study the Fanconi anemia associated protein 20 (FAAP20)-ubiquitin complex within the Fanconi Anemia (FA) complexes required for efficient repair of DNA interstrand crosslinks (ICLs), a process that is mediated by the ubiquitin-binding zinc finger (UBZ) domain of FAAP20. Unexpectedly, we show that the FAAP20-ubiquitin interaction extends beyond the compact UBZ module and is accompanied by transforming the disordered C-terminal tail of FAAP20 into a rigid &beta-loop, with the invariant C-terminal tryptophan (W180 of human FAAP20) emanating toward I44 of ubiquitin for enhanced binding. Accordingly, alanine substitution of the absolutely conserved C-terminal tryptophan residue of FAAP20 abolishes ubiquitin binding and impairs FA core complex-mediated ICL repair in vivo.

Reliable detection and unambiguous assignment of intermolecular NOEs is essential to NMR-based structure determination of protein complexes. The development of 4-D omit NOE spectroscopy in this thesis overcomes many limitations of conventional 3-D half-filtered experiments to allow for reliable detection and unambiguous assignment of intermolecular NOEs of heterodimeric complexes and homooligomeric complexes. These advantages render such a strategy particularly attractive for structural studies of protein complexes by biomolecular NMR.

Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint.

Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the Xenopus egg extract system, our laboratory has previously demonstrated that the RING domain-containing ubiquitin E3 ligase Xnf7 can inhibit Xenopus APC/C activity. In the thesis, we have identified TRIM39 as an Xnf7-related human regulator of the APC/C. Our study showed that TRIM39 restrains the ability of the APC/C to ubiquitylate Cyclin B in vitro and attenuates the degradation of Cyclin B and geminin when TRIM39 is incubated in cell lysates. Notably, it has been reported that TRIM39 activity is responsible for the accumulation of the Bax-interacting protein (and activator) MOAP-1 following etoposide-induced DNA damage. Our data indicated that MOAP-1 is a novel APC/C substrate, and that the ligase activity of TRIM39 appears to be essential for preventing its degradation. We further demonstrated that decreased levels of the APC/C activator Cdh1 induces MOAP-1 protein accumulation, thereby promoting DNA damage-induced apoptosis in 293T, PC3 and H1299 cells. This study illustrates a potential function for the APC/C in DNA damage induced apoptosis and also demonstrates that TRIM39 regulates both the cell cycle and apoptosis via APC/C inhibition.

To extend our observations regarding the role for TRIM39 in APC/C regulation, we investigated effects on the cell cycle via real-time imaging microscopy. We found cells arrest at G1/S in TRIM39 depleted RPE cells, a cell line which is commonly used for cell cycle analysis. This arrest phenotype is not observed in 293T, PC3 and H1299 cells which bear mutant p53 alleles. Further analysis showed that TRIM39 depleted RPE cells upregulate many genes that function downstream of p53 activity, such as the cdk inhibitor p21--thus, arresting cells at G1/S and reducing proliferation. The reduced growth can be rescued by p53 knockdown. Mechanistically, TRIM39 interacts with p53 and promotes destruction of p53 by ubiquitylation. This ubiquitylation is independent of the activity of the most intensively studied p53-directed E3 ligase, MDM2; depletion of both MDM2 and TRIM39 has a synergistic effect on p53 accumulation. This elevated p53 leads to more apoptosis in cancer cells bearing wildtype p53. Consequently, TRIM39 depletion might be employed as a combination treatment with MDM2 inhibitor, such as nutlin-3a, to stimulate tumor cell death.

In the thesis, we have found TRIM39 inhibits both the APC/C and p53. Both are essential regulators of cell cycle and apoptosis. Moreover, we have determined that the inhibitory activity of TRIM39 requires its E3 ligase activity. Future experiments will be directed towards investigating how TRIM39 protein stability and ligase activity are regulated to understand more fully the physiological situations in which TRIM39 is able to exert its ability to modulate the cell cycle and apoptosis. I will also discuss some preliminary data regarding changes in TRIM39 ligase activity induced by Chk1 and changes in TRIM39 protein abundance regulated by polo-like kinase 1(Plk1). Chk1 and Plk1 are essential kinases for cell cycle checkpoint and progression. Connecting Chk1 and Plk1 to TRIM39 may provide a more thorough understanding of TRIM39's ability to control the APC/C inhibition and p53 ubiquitylation in response to cell cycle or cell damage cues. Since the APC/C and p53 both can regulate cell cycle and apoptosis, further investigations into the involvement of TRIM39 in the life-or-death decision will be of great interest.