Neuropathological Outcomes of Traumatic Brain Injury and Alcohol Use in Males and Females: Studies Using Pre-Clinical Rodent and Clinical Human Specimens.

Traumatic brain injury (TBI) and alcohol misuse are inextricably linked and can increase the risk for development of neurodegenerative diseases, particularly in military veterans and contact sport athletes. Proteinopathy (defects in protein degradation) is considered an underlying factor in neurodegenerative diseases. Whether it contributes to TBI/alcohol-mediated neurodegeneration is unexplored, however. Our recent studies have identified ISGylation, a conjugated form of ISG15 (Interferon-Stimulated Gene 15) and inducer of proteinopathy, as a potential mechanistic link underlying TBI-mediated neurodegeneration and proteinopathy in veterans. In the current study, a rat model of combined TBI and alcohol use was utilized to investigate the same relationship. Here, we report sustained induction of Interferon ß (IFNß), changes in TAR DNA Binding 43 (TDP-43) ISGylation levels, TDP-43 proteinopathy (C-terminal fragmentation [CTF]), and neurodegeneration in the ventral horns of the lumbar spinal cords (LSCs) and/or motor cortices (MCs) of female rats post-TBI in a time-dependent manner. In males, these findings mostly remained non-significant, although moderate alcohol use appears to decrease neurodegeneration in males (but not females) post-TBI. We, however, do not claim that moderate alcohol consumption is beneficial for preventing TBI-mediated neurodegeneration. We have previously demonstrated that ISGylation is increased in the LSCs of veterans with TBI/ALS (amyotrophic lateral sclerosis). Here, we show increased ISGylation of TDP-43 in the LSCs of TBI/ALS-afflicted female veterans compared with male veterans. Knowing that ISGylation induces proteinopathy, we suggest targeting ISGylation may prevent proteinopathy-mediated neurodegeneration post-TBI, particularly in women; however, causal studies are required to confirm this claim.

Evidence for the Deregulation of Protein Turnover Pathways in Atm-Deficient Mouse Cerebellum: An Organotypic Study.

Interferon-stimulated gene 15 (ISG15), an antagonist of the ubiquitin pathway, is elevated in cells and brain tissues obtained from ataxia telangiectasia (A-T) patients. Previous studies reveal that an elevated ISG15 pathway inhibits ubiquitin-dependent protein degradation, leading to activation of basal autophagy as a compensatory mechanism for protein turnover in A-T cells. Also, genotoxic stress (ultraviolet [UV] radiation) deregulates autophagy and induces aberrant degradation of ubiquitylated proteins in A-T cells. In the current study, we show that, as in A-T cells, ISG15 protein expression is elevated in cerebellums and various other tissues obtained from Atm-compromised mice in an Atm-allele-dependent manner (Atm+/+ < Atm+/- < Atm-/-). Notably, in cerebellums, the brain part primarily affected in A-T, levels of ISG15 were significantly greater (3-fold higher) than cerebrums obtained from the same set of mice. Moreover, as in A-T cell culture, UV induces aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient, but not in Atm-proficient, cerebellar brain slices grown in culture. Thus, the ex vivo organotypic A-T mouse brain culture model mimics that of an A-T human cell culture model and could be useful for studying the role of ISG15-dependent proteinopathy in cerebellar neurodegeneration, a hallmark of A-T in humans.

ISG15: A double edged sword in cancer.

Interferon-Stimulated Gene 15 (ISG15) transcript is aberrantly expressed in most human malignancies, suggesting that it has a protumor function. However, at the protein level ISG15 has both protumor and immunomodulatory antitumor functions. Therapeutic strategies to maximize the latter may benefit cancer patients overexpressing the ISG15 pathway.

Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective.

Interferon-Stimulated Gene 15 (ISG15), an antagonist of the canonical ubiquitin pathway, is frequently overexpressed in various cancers. In cancer cells, ISG15 is detected as free (intracellular) and conjugated to cellular proteins (ISGylation). Free ISG15 is also secreted into the extracellular milieu. ISGylation has protumor functions and extracellular free ISG15 has immunomodulatory properties in vitro. Therefore, whether ISG15 is a tumor suppressor or tumor promoter in vivo remains controversial. The current study aimed to clarify the role of free ISG15 in tumorigenesis. Breast cancer cells stably expressing control, ISG15, and UbcH8 (ISG15-specific E2 ligase) shRNAs were used to assess the immunoregulatory and antitumor function of free ISG15 in cell culture (in vitro) and in nude mice (in vivo). We show that extracellular free ISG15 suppresses breast tumor growth and increases NK cell infiltration into xenografted breast tumors in nude mice, and intracellular free ISG15 enhances major histocompatibility complex (MHC) class I surface expression in breast cancer cells. We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo. These findings provides the basis for developing strategies to increase systemic levels of free ISG15 to treat cancer patients overexpressing the ISG15 pathway.

Suppression of the macrophage proteasome by ethanol impairs MHC class I antigen processing and presentation.

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

ISG15 deregulates autophagy in genotoxin-treated ataxia telangiectasia cells.

Ataxia-telangiectasia (A-T) is a cerebellar neurodegenerative disorder; however, the basis for the neurodegeneration in A-T is not well established. Lesions in the ubiquitin and autophagy pathways are speculated to contribute to the neurodegeneration in other neurological diseases and may have a role in A-T neurodegeneration. Our recent studies revealed that the constitutively elevated ISG15 pathway impairs targeted proteasome-mediated protein degradation in A-T cells. Here, we demonstrate that the basal autophagy pathway is activated in the ubiquitin pathway-compromised A-T cells. We also show that genotoxic stress triggers aberrant degradation of the proteasome and autophagy substrates (autophagic flux) in A-T cells. Inhibition of autophagy at an early stage using 3-methyladenine blocked UV-induced autophagic flux in A-T cells. On the other hand, bafilomycin A1, which inhibits autophagy at a late stage, failed to block UV-induced autophagic flux, suggesting that overinduction of autophagy may underlie aberrant autophagic flux in A-T cells. The ISG15-specific shRNA that restored proteasome function restores autophagic function in A-T cells. These findings suggest that autophagy compensates for the ISG15-dependent ablation of proteasome-mediated protein degradation in A-T cells. Genotoxic stress overactivates this compensatory mechanism, triggering aberrant autophagic flux in A-T cells. Supporting the model, we show that autophagy is activated in the brain tissues of human A-T patients. This highlights a plausible causal contribution of a novel "ISG15 proteinopathy" in A-T neuronal cell death.

ISG15 disrupts cytoskeletal architecture and promotes motility in human breast cancer cells.

The interferon-stimulated gene 15 (ISG15) pathway is highly elevated in breast cancer; however, very little is known about how the ISG15 pathway contributes to breast tumorigenesis. In the current study, using the gene disruption approach, we demonstrate that both ISG15 and UbcH8 (ISG15-specific conjugating enzyme) disrupt F-actin architecture and formation of focal adhesions in ZR-75-1 breast cancer cells. In addition, ISG15 and UbcH8 promote breast cancer cell migration. We also demonstrate that ISG15 inhibits ubiquitin/26S proteasome-mediated turnover of proteins implicated in tumor cell motility, invasion and metastasis. Together, our results suggest that the aberrant activation of the ISG15 pathway confers a motile phenotype to breast cancer cells by disrupting cell architecture and stabilizing proteins involved in cell motility, invasion and metastasis. Because the cellular architecture is conserved and the ISG15 pathway is constitutively activated in tumor cells of different lineages, it is reasonable to assume that our observations in breast cancer must hold true for many other tumors.

A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway.

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

A ubiquitin-proteasome pathway for the repair of topoisomerase I-DNA covalent complexes.

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.

ISG15 as a novel tumor biomarker for drug sensitivity.

Tumor cells are known to exhibit highly varied sensitivity to camptothecins (CPT; e.g., irinotecan and topotecan). However, the factors that determine CPT sensitivity/resistance are largely unknown. Recent studies have shown that the ubiquitin-like protein, IFN-stimulated gene 15 (ISG15), which is highly elevated in many human cancers and tumor cell lines, antagonizes the ubiquitin/proteasome pathway. In the present study, we show that ISG15 is a determinant for CPT sensitivity/resistance possibly through its effect on proteasome-mediated repair of topoisomerase I (TOP1)-DNA covalent complexes. First, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 (major E2 for ISG15) in breast cancer ZR-75-1 cells decreased CPT sensitivity, suggesting that ISG15 overexpression in tumors could be a factor affecting intrinsic CPT sensitivity in tumor cells. Second, the level of ISG15 was found to be significantly reduced in several tumor cells selected for resistance to CPT, suggesting that altered ISG15 regulation could be a significant determinant for acquired CPT resistance. Parallel to reduced CPT sensitivity, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 in ZR-75-1 cells resulted in increased proteasomal degradation of CPT-induced TOP1-DNA covalent complexes. Taken together, these results suggest that ISG15, which interferes with proteasome-mediated repair of TOP1-DNA covalent complexes, is a potential tumor biomarker for CPT sensitivity.

Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway.

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/degradation.

Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death.

Reactive oxygen species modify DNA, generating various DNA lesions including modified bases such as 8-oxoguanine (8-oxoG). These base-modified DNA lesions have been shown to trap DNA topoisomerase I (TOP1) into covalent cleavage complexes. In this study, we have investigated the role of TOP1 in hydrogen peroxide toxicity. We showed that ectopic expression of TOP1 in Saccharomyces cerevisiae conferred sensitivity to hydrogen peroxide, and this sensitivity was dependent on RAD9 checkpoint function. Moreover, in the mammalian cell culture system, hydrogen peroxide-induced growth inhibition and apoptosis were shown to be partly TOP1-dependent as evidenced by a specific increase in resistance to hydrogen peroxide in TOP1-deficient P388/CPT45 murine leukemia cells as compared with their TOP1-proficient parental cell line P388. In addition, hydrogen peroxide was shown to induce TOP1-DNA cross-links. These results support a model in which hydrogen peroxide promotes the trapping of TOP1 on oxidative DNA lesions to form TOP1-DNA cleavage complexes that contribute to hydrogen peroxide toxicity.

Characterization of ARC-111 as a novel topoisomerase I-targeting anticancer drug.

8,9-Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6] naphthyridin-6-one (ARC-111, topovale) is a new synthetic antitumor agent. In the current study, we show that ARC-111 is highly potent in scid mice carrying human tumor xenografts. ARC-111 was shown to be as active as camptothecin (CPT)-11 in the HCT-8 colon tumor model, and compared favorably with CPT-11 and topotecan in the SKNEP anaplastic Wilms' tumor model. In tissue culture models, ARC-111 exhibited low nM cytotoxicity against a panel of cancer cells. ARC-111 cytotoxicity as well as ARC-111-induced apoptosis was reduced >100-fold in CPT-resistant topoisomerase I (TOP1)-deficient P388/CPT45 cells as compared with P388 cells. Similarly, ARC-111 cytotoxicity was greatly reduced in CPT-resistant CPT-K5 and U937/CR cells, which express CPT-resistant mutant TOP1, suggesting that the cytotoxic target of ARC-111 is TOP1. Indeed, ARC-111, like CPT, was shown to induce reversible TOP1 cleavage complexes in tumor cells as evidenced by specific reduction of the TOP1 immunoreactive band in a band depletion assay, as well as elevation of small ubiquitin modifier-TOP1 conjugate levels and activation of 26S proteasome-mediated degradation of TOP1. Unlike CPT, ARC-111 is not a substrate for the ATP-binding cassette transporter breast cancer resistance protein. In addition, ARC-111 cytotoxicity was not significantly reduced in the presence of human serum albumin. These results suggest that ARC-111 is a promising new TOP1-targeting antitumor drug with a different drug resistance profile than CPT.

The topoisomerase IIbeta circular clamp arrests transcription and signals a 26S proteasome pathway.

It has been proposed that the topoisomerase II (TOP2)beta-DNA covalent complex arrests transcription and triggers 26S proteasome-mediated degradation of TOP2beta. It is unclear whether the initial trigger for proteasomal degradation is due to DNA damage or transcriptional arrest. In the current study we show that the TOP2 catalytic inhibitor 4,4-(2,3-butanediyl)-bis(2,6-piperazinedione) (ICRF-193), which traps TOP2 into a circular clamp rather than the TOP2-DNA covalent complex, can also arrest transcription. Arrest of transcription, which is TOP2beta-dependent, is accompanied by proteasomal degradation of TOP2beta. Different from TOP2 poisons and other DNA-damaging agents, ICRF-193 did not induce proteasomal degradation of the large subunit of RNA polymerase II. These results suggest that proteasomal degradation of TOP2beta induced by the TOP2-DNA covalent complex or the TOP2 circular clamp is due to transcriptional arrest but not DNA damage. By contrast, degradation of the large subunit of RNA polymerase II is due to a DNA-damage signal.

Transcription-dependent degradation of topoisomerase I-DNA covalent complexes.

Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR.