Health Care Resource Use Among Patients with Advanced Non-Small Cell Lung Cancer in Japan, 2017-2019.

Background: First-line immune checkpoint inhibitor (ICI) monotherapy for advanced non-small cell lung cancer (NSCLC) was introduced in Japan in February 2017. Limited information is available since that time regarding health care resource use for NSCLC in Japan, where the hospitalization burden is high. Objective: We evaluated health care resource use from first- through third-line systemic anticancer therapy for patients with advanced NSCLC included in a multicenter, retrospective chart review study. Methods: Eligible patients were aged 20 years or older with unresectable locally advanced/metastatic NSCLC with no known actionable genomic alteration who initiated first-line systemic anticancer therapy from July 1, 2017, to December 20, 2018, at 23 Japanese hospitals. We calculated the percentage of patients with a record of each resource used, the total number of each resource, and the resource use per 100 patient-weeks of follow-up from initiation of first-, second-, and third-line therapy, overall and by the 3 most common regimen categories, namely, ICI monotherapy, platinum-doublet chemotherapy (without concomitant ICI), and nonplatinum cytotoxic regimens (nonplatinum). Study follow-up ended September 30, 2019. Results: Among 1208 patients (median age = 70 years; 975 [81%] men), 463 patients (38%) received ICI monotherapy, 647 (54%) received platinum-doublet chemotherapy, and 98 (8%) received nonplatinum regimens as first-line therapy. During the study, 621 (51%) patients initiated second-line, and 281 (23%) initiated third-line therapy. The majority of patients experienced ≥1 hospitalization (76%-94%) and ≥1 outpatient visit (85%-90%) during each therapy line. The number of hospitalizations increased from 6.5 per 100 patient-weeks in first-line to 8.0 per 100 patient-weeks in third-line. During first-line therapy, the number of hospitalizations per 100 patient-weeks were 4.8, 8.4, and 6.5 for patients receiving ICI monotherapy, platinum-doublet chemotherapy, and nonplatinum regimens, respectively, and the percentages of hospitalizations categorized as attributable to NSCLC treatment administration (no surgery, procedure, treatment of metastasis, or palliative lung radiation) were 64%, 77%, and 73%, respectively. The number of outpatient visits increased from 43.0 per 100 patient-weeks in first-line to 51.4 per 100 patient-weeks in third-line therapy. During first-line therapy, outpatient visits per 100 patient-weeks were 41.0, 46.7, and 33.0 for patients receiving ICI monotherapy, platinum-doublet chemotherapy, and nonplatinum regimens, respectively, and the percentages of outpatient visits for infusion therapy were 48%, 34%, and 36%, respectively. Conclusions: The results of this study, although solely descriptive, showed differing patterns of health care resource use during first-line therapy among the 3 common systemic anticancer therapy regimens for advanced NSCLC in Japan and suggest that further research is needed to investigate these apparent differences by treatment regimen.

First-Line Pembrolizumab Monotherapy for Advanced NSCLC With Programmed Death-Ligand 1 Expression Greater Than or Equal to 50%: Real-World Study Including Older Patients in Japan.

Introduction: Pembrolizumab became available in Japan in February 2017 for first-line monotherapy of unresectable advanced and metastatic NSCLC with programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) greater than or equal to 50%. This retrospective chart review study aimed to describe real-world clinical outcomes of first-line pembrolizumab monotherapy, including for patients 75 years or older, who are under-represented in clinical trials. Methods: We identified patients (≥20 y old) at 23 sites initiating first-line pembrolizumab monotherapy from July 1, 2017, to December 20, 2018, for stages IIIB, IIIC, and IV NSCLC with PD-L1 TPS greater than or equal to 50% and Eastern Cooperative Oncology Group performance status of 0 to 2 or unknown. Patients with actionable genomic alterations (EGFR, ALK, ROS1, BRAF) and clinical trial participants were excluded. Time-to-event outcomes were estimated using Kaplan-Meier, with data cutoff on September 30, 2019. Results: Of 441 eligible patients (78% men), 303 (69%) were younger than 75 years and 138 (31%) were 75 years or older; median age was 70 years. With median follow-up of 13.5 months, median overall survival (OS) was not reached (NR); 12- and 24-month OS rates were 72% and 58%, respectively. For ages younger than 75 and 75 years or older, median OS was NR and 23.5 months (95% confidence interval: 16.2-NR), respectively; 12-month OS rates were 74% and 67% and 24-month OS rates were 62% and 48%, respectively. Median real-world progression-free survival was similar in the two age groups (10.1 and 9.5 mo, respectively), as was median real-world time on treatment with pembrolizumab (5.7 and 5.6 mo). Conclusions: These findings complement clinical trial results, adding real-world evidence supporting benefits of first-line pembrolizumab monotherapy for advanced NSCLC with PD-L1 TPS greater than or equal to 50%, including for patients 75 years or older.

Real-World Treatments and Clinical Outcomes in Advanced NSCLC without Actionable Mutations after Introduction of Immunotherapy in Japan.

The aims of this study were to describe systemic treatment patterns and clinical outcomes for unresectable advanced/metastatic non-small-cell lung cancer (NSCLC) by first-line regimen type in real-world clinical settings in Japan after the introduction of first-line immune checkpoint inhibitor (ICI) monotherapy in 2017. Using retrospective chart review at 23 study sites, we identified patients ≥20 years old initiating first-line systemic therapy from 1 July 2017 to 20 December 2018, for unresectable stage IIIB/C or IV NSCLC; the data cutoff was 30 September 2019. Eligible patients had recorded programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) and no known actionable EGFR/ALK/ROS1/BRAF genomic alteration. Kaplan-Meier method was used to determine time-to-event endpoints. Of 1208 patients, 647 patients (54%) received platinum doublet, 463 (38%) received ICI monotherapy, and 98 (8%) received nonplatinum cytotoxic regimen as first-line therapy. PD-L1 TPS was ≥50%, 1−49% and <1% for 44%, 30%, and 25% of patients, respectively. Most patients with PD-L1 TPS ≥50% received ICI monotherapy (453/529; 86%). Excluding 26 patients with ECOG performance status of 3−4 from outcome analyses, the median patient follow-up was 11.3 months. With first-line platinum doublet, ICI monotherapy, and nonplatinum cytotoxic regimens, median overall survival (OS) was 16.3 months (95% CI, 14.0−20.1 months), not reached, and 14.4 months (95% CI, 10.3−21.2 months), respectively; 24-month OS was 40%, 58%, and 31%, respectively. Differences in OS relative to historical cohort data reported in Japan are consistent with improvement over time in real-world clinical outcomes for advanced NSCLC.

Real-world outcomes of pembrolizumab monotherapy in non-small cell lung cancer in Japan: A post-marketing surveillance.

This post-marketing surveillance (PMS) was initiated in Japan to identify factors affecting the safety and effectiveness of pembrolizumab monotherapy in patients with advanced non-small cell lung cancer (NSCLC) with programmed cell death ligand-1 (PD-L1) expression. This PMS was conducted from December 2016 to June 2019 at 717 centers across Japan. Patients with unresectable advanced/recurrent NSCLC who received pembrolizumab monotherapy as first-line (1L) treatment for PD-L1-expressing tumors (Tumor Proportion Score [TPS] ≥ 50%) or second-line or later (2L+) treatment for tumors with PD-L1 TPS ≥ 1% were enrolled and followed up for 1 year. Of 2805 registered patients, 2740 and 2400 comprised the safety and effectiveness analysis sets, respectively. The median age (range) was 69 (27-92) years; 55.7% and 29.2% of patients experienced treatment-related adverse events and adverse events of special interest (AEOSIs), respectively. More common AEOSIs included interstitial lung disease, endocrine disorders, liver dysfunction, colitis/severe diarrhea, infusion reactions, and severe skin disorders. The frequency of experiencing ≥2 AEOSIs was low (1L, 6.5%; 2L+, 2.8%). Most AEOSIs occurred within 150 days after initiation of pembrolizumab monotherapy. At 1-year follow-up, the objective response rate was 39.2% (1L, 51.5%; 2L+, 30.0%). In conclusion, the 1-year safety and effectiveness of pembrolizumab monotherapy in patients with unresectable advanced/recurrent NSCLC as 1L treatment for tumors with PD-L1 TPS ≥ 50% and 2L+ treatment for tumors with PD-L1 TPS ≥ 1% were similar to those reported in phase 2/3 trials.

The role of NUB1 in α-synuclein degradation in Lewy body disease model mice.

Abnormal α-synuclein is deposited in neuronal cytoplasmic inclusions and presynapses in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NUB1 is accumulated in these specific regions together with abnormal α-synuclein and that NUB1 is able to inhibit α-synuclein aggregation in cultured cells. We therefore created transgenic (Tg) mice expressing both NUB1 and abnormal α-synuclein to investigate the role of NUB1 on degradation of abnormal α-synuclein in vivo. Immunohistochemical and biochemical studies confirmed that NUB1 was over-expressed in neurons of mice expressing NUB1 (NUB1 Tg), and both NUB1 and abnormal α-synuclein (double Tg). NUB1 levels were increased by 4.7-fold in NUB1 Tg mice compared with wild type mice. Unexpectedly, normal and abnormal α-synuclein levels were unchanged between abnormal α-synuclein Tg mice (Lewy body disease model mice) and double Tg mice, and pathological observations were almost similar between them. Finally, we found that the levels of insoluble α-synuclein were lower and those of some chaperone molecules were higher in double Tg mice compared with abnormal α-synuclein Tg mice. These results suggest that increased levels of NUB1 play a potential role in degradation of detergent-insoluble α-synuclein in vivo, although it is insufficient to degrade abnormal α-synuclein in Lewy body disease model mice.

Role of Ser129 phosphorylation of α-synuclein in melanoma cells.

α-Synuclein, a protein central to Parkinson's disease, is frequently expressed in melanoma tissues, but not in non-melanocytic cutaneous carcinoma and normal skin. Thus, α-synuclein is not only related to Parkinson's disease, but also to melanoma. Recently, epidemiologists reported co-occurrence of melanoma and Parkinson's disease in patients, suggesting that these diseases could share common pathogenetic components and that α-synuclein might be one of these. In Parkinson's disease, phosphorylation of α-synuclein at Ser129 plays an important role in the pathobiology. However, its role in melanoma is not known. Here, we show the biological relevance of Ser129 phosphorylation in human melanoma cells. First, we have identified an antibody that reacts with Ser129-unphosphorylated α-synuclein but not with Ser129-phosphorylated α-synuclein. Using this and other antibodies to α-synuclein, we investigated the role of Ser129 phosphorylation in human melanoma SK-MEL28 and SK-MEL5 cells. Our immunofluorescence microscopy showed that the Ser129-phosphorylated form, but not the Ser129-unphosphorylated form, of α-synuclein localizes to dot-like structures at the cell surface and the extracellular space. Furthermore, immuno-electron microscopy showed that the melanoma cells release microvesicles in which Ser129-phosphorylated α-synuclein localizes to the vesicular membrane. Taken together, our studies suggest that the phosphorylation of Ser129 leads to the cell surface translocation of α-synuclein along the microtubule network and its subsequent vesicular release in melanoma cells.

Negative regulation of NEDD8 conjugation pathway by novel molecules and agents for anticancer therapy.

Tumor cells frequently promote the dysregulation of the cell cycle and escape from apoptotic cell death triggered by a number of cellular stresses. Programmed proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway is crucial for homeostasis of numerous biological processes. Disruption of this system is one of the factors that promote aberrant cell-proliferation. The small ubiquitin-like protein, NEDD8, has been identified as a fundamental regulator of the activity of the E3 ubiquitin ligases called the SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) which control a final step in ubiquitination of diverse substrates associated with cancer biology. The ubiquitin ligase activity of the SCF complex requires NEDD8 to covalently bind to cullins. To a large extent, exploring the negative regulation system of the NEDD8 pathway is expected to lead to the development of novel anticancer targets. This review focuses on the NEDD8 negative regulation system including chemical compounds such as MLN4924 and protein molecules (e.g. COP9 signalosome, CAND1, inactive mutant of Ubc12 and NUB1/NUB1L) and clarifies possible strategies for targeting the NEDD8 cascade in cancer cells.

Ubiquitin-related proteins in neuronal and glial intranuclear inclusions in intranuclear inclusion body disease.

Recent studies have shown that eosinophilic intranuclear inclusions (INI) in the brain of patients with intranuclear inclusion body disease (INIBD) are immunopositive for ubiquitin and ubiquitin-related proteins (URP). However, the extent and frequency of URP-immunoreactive inclusions in INIBD are uncertain. We immunohistochemically examined the brain, spinal cord and dorsal root ganglia from five patients with INIBD, using a virtual slide system with sequential staining of the same sections with hematoxylin and eosin and by immunolabeling with antibodies against ubiquitin and URP (NEDD8, NUB1, SUMO-1 and SUMO-2). Intranuclear inclusions were widely distributed in neurons and glial cells in all the cases. Sequential staining revealed that 100% of INI in neurons and glial cells were positive for ubiquitin. Moreover, the majority or a significant proportion of INI were positive for NEDD8, NUB1, SUMO-1 and SUMO-2. However, the proportions of NEDD8-, NUB1- and SUMO-1-positive inclusions were significantly higher in neurons than in glial cells (P < 0.05). These findings suggest that proteins related to ubiquitination and proteasomal degradation are involved in the formation of INI in INIBD.

A simple and sensitive method for the demonstration of norepinephrine-storing adrenomedullary chromaffin cells.

The medulla of the adrenal gland is a neuroendocrine tissue in which catecholamine-storing chromaffin cells exist. The chromaffin cells are derived from neural crest cells and distinctly differentiated into two types of cells, epinephrine (E) (adrenaline)-storing and norepinephrine (NE) (noradrenaline)-storing cells. Using histochemical or immunostaining methods, the two types of chromaffin cells have been differentially distinguished. However, difficulties and/or drawbacks of the procedures have somewhat restricted the progress of research in differential functions of E-storing and NE-storing cells. Here, we show a new method for the differential demonstration of these two cell types. We found that mouse and rat adrenomedullary cells are heterogeneously stained with Harris hematoxylin after treatment with citrate buffer at pH 6. The cell clusters stained with hematoxylin were positive for tyrosine hydroxylase, which is an enzyme involved in catecholamine biosynthesis. Furthermore, the cell clusters were negative for phenylethanolamine-N-methyl transferase, which is an enzyme responsible for the conversion from NE to E and expresses in E-storing chromaffin cells. Moreover, we found that the cell clusters stained with hematoxylin can also be stained with nitroblue tetrazolium at pH 11, using Hopsu and Mäkinen's method by which NE-storing chromaffin cells are stained. These observations indicate that the cytoplasm of NE-storing chromaffin cells is specifically stained with hematoxylin after treatment with citrate buffer at pH 6. This method will allow us to facilitate cell-type specific research of chromaffin cells. Indeed, this method revealed that α-synuclein selectively expresses in E-storing chromaffin cells, but not in NE-storing chromaffin cells.

Inhibition of NEDD8-conjugation pathway by novel molecules: potential approaches to anticancer therapy.

Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell-cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8-conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.

Immunohistochemical analysis of Marinesco bodies, using antibodies against proteins implicated in the ubiquitin-proteasome system, autophagy and aggresome formation.

Marinesco bodies (MBs) are spherical eosinophilic intranuclear inclusions in pigmented neurons in the substantia nigra and locus ceruleus. Previous immunohistochemical studies have shown that MBs are positive for ubiquitin, p62 and SUMO-1, suggesting the involvement of ubiquitination and related proteins in the formation or disaggregation of MBs. However, the involvement is not thoroughly understood. Therefore, we immunohistochemically examined the midbrain from five control subjects ranged from 53 to 84 years old. MBs were positive for various proteins implicated in the ubiquitin-proteasome system (ubiquitin, p62, EDD1, NEDD8, NUB1, SUMO-1 and SUMO-2), aggresome formation (HDAC6) and autophagy (ubiquitin, p62, LC3, GABARAP and GATE-16). These findings suggest that proteins related to ubiquitination, proteasomal degradation and autophagy are involved in the formation or disaggregation of MBs.

Pathobiochemical effect of acylated steryl-ß-glucoside on aggregation and cytotoxicity of α-synuclein.

Cycad seed consumption by the native islanders of Guam is frequently associated with high rates of amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC); furthermore, accompanying pathological examination often exhibits α-synuclein inclusions in the neurons of the affected brain. Acylated steryl-ß-glucoside (ASG) contained in cycad seeds is considered as causative environmental risk factor. We aimed to investigate whether ASG influences aggregation and cell toxicity of α-synuclein. To understand whether ASG is a causative factor in the development of ALS/PDC, soybean-derived ASG was tested for its effect on in vitro aggregation of α-synuclein using Thioflavin-T. ASG was also tested to determine whether it modulates α-synuclein cytotoxicity in yeast cells. In addition, we determined whether an interaction between ASG and α-synuclein occurs in the plasma membrane or cytoplasm using three factors: GM1 ganglioside, small unilamellar vesicles, and ATP. In the present study, we found that ASG-mediated acceleration of α-synuclein aggregation is influenced by the presence of ATP, but not by the presence of GM1. ASG accelerated the α-synuclein aggregation in the cytoplasm. ASG also enhanced α-synuclein-induced cytotoxicity in yeast cells. This study demonstrated that ASG directly enhances aggregation and cytotoxicity of α-synuclein, which are often observed in patients with ALS/PDC. These results, using assays that replicate cytoplasmic conditions, are consistent with the molecular mechanism that cytotoxicity is caused by intracellular α-synuclein fibril formation in neuronal cells.

Improved immunodetection of endogenous α-synuclein.

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.

Synphilin-1-binding protein NUB1 is colocalized with nonfibrillar, proteinase K-resistant α-synuclein in presynapses in Lewy body disease.

α-Synuclein is a major component of Lewy bodies in Parkinson disease (PD) and dementia with Lewy bodies (DLB). We recently showed that abnormal α-synuclein with resistance to proteinase K (PK) is deposited at presynapses of distinct brain anatomic regions from the early stages of PD and DLB. NUB1, a synphilin-1-binding protein, also accumulates in Lewy bodies, but it is not known whether abnormal α-synuclein is associated with NUB1. Here, we demonstrate that, in the brain of patients with PD and DLB, NUB1 accumulates in the presynapses in the hippocampus, cerebral neocortex, and substantia nigra in which PK-resistant α-synuclein is deposited. Endogenous NUB1 also accumulated with PK-resistant α-synuclein in the presynapses of transgenic mice that express human α-synuclein with an A53T mutation. Immunoelectron microscopy showed that NUB1 is localized to presynaptic nerve terminals where no abnormal filaments are seen. Biochemical analyses showed that NUB1 coexists with abnormal α-synuclein in the brain of DLB patients. These findings suggest that NUB1 along with abnormal α-synuclein is involved in the pathogenesis of Lewy body disease.

Cytoplasmic relocation of Daxx induced by Ro52 and FLASH.

The RING-finger protein Ro52/TRIM21 is known to be an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren's syndrome and systemic lupus erythematosus. We recently showed that Ro52 is an E3 ubiquitin ligase and localizes to cytoplasmic bodies that are highly motile along the microtubule network. To expand our knowledge of Ro52, we searched partners co-operating with Ro52. We performed a yeast two-hybrid screening of a human brain cDNA library with Ro52 as bait. This screening identified several genes encoding Ro52-interacting proteins, including the apoptosis-related proteins, Daxx and FLASH. Further yeast two-hybrid assays revealed that Daxx binds to the B30.2 domain of Ro52 and that FLASH binds to coiled-coil domains of Ro52 through its death-effector domain-recruiting domain. These results suggest that Ro52, Daxx, and FLASH form heteromeric protein complexes. Indeed, this was supported by results of immunoprecipitation experiments in which we found that Daxx is co-immunoprecipitated with Ro52 in the presence of overexpressed FLASH. Importantly, our fluorescence microscopy revealed that, although Daxx is predominantly located in the nucleus, overexpression of both Ro52 and FLASH leads to relocation of Daxx into the cytoplasm. Thus, Ro52 seems to co-operate with FLASH to induce cytoplasmic localization of Daxx in cells.

Downregulation of active IKK beta by Ro52-mediated autophagy.

Upon activation, NF-kappaB translocates into the nucleus and initiates many biological events. This NF-kappaB signaling is mainly induced by the protein kinase IKK beta. Early in this signaling pathway, IKK beta is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1). In cells expressing Tax protein, IKK beta is persistently phosphorylated, which chronically activates NF-kappaB signaling. But the active IKK beta is conjugated with a monoubiquitin by the E3 ubiquitin ligase Ro52, and the IKK beta-induced NF-kappaB signaling is downregulated. However, the mechanism of the downregulation has been unknown. Here, we show that Ro52-mediated monoubiquitination is involved in the subcellular translocation of active IKK beta to autophagosomes. Furthermore, using reporter assays, we show that Ro52 suppresses IKK beta-induced NF-kappaB signaling and that this suppression is blocked by an autophagy inhibitor. These results suggest that Ro52-mediated monoubiquitination plays a critical role in the downregulation of active IKK beta through autophagy.

Parkinson's disease-related protein, alpha-synuclein, in malignant melanoma.

BACKGROUND: Melanoma is the major cause of skin cancer death worldwide. Parkinson's disease is a neurodegenerative disorder that is caused by mutation of alpha-synuclein or other genes. Importantly, epidemiological studies have reported co-occurrence of melanoma and Parkinson's disease, suggesting that these two diseases could share common genetic components. METHODOLOGY/PRINCIPAL FINDINGS: Recently, we found that human melanoma cell lines highly express alpha-synuclein, whereas the protein is undetectable in the non-melanoma cancer cell lines tested. To investigate the expression of alpha-synuclein in human melanoma tissues, we immunostained sections of melanoma, nevus, non-melanocytic cutaneous carcinoma, and normal skin. alpha-Synuclein was positively detected in 86% of the primary and 85% of the metastatic melanoma sections, as well as in 89% of nevus sections. However, alpha-synuclein was undetectable in non-melanocytic cutaneous carcinoma and normal skin. CONCLUSIONS/SIGNIFICANCE: The Parkinson's disease-related protein, alpha-synuclein, is expressed in both malignant and benign melanocytic lesions, such as melanomas and nevi. Although alpha-synuclein cannot be used to distinguish between malignant and benign melanocytic skin lesions, it might be a useful biomarker for the diagnosis of metastatic melanoma.

TRIM9, a novel brain-specific E3 ubiquitin ligase, is repressed in the brain of Parkinson's disease and dementia with Lewy bodies.

TRIM family proteins are involved in a broad range of biological processes, and their alteration results in many diverse pathological conditions found in genetic diseases, viral infections, and cancers. However, the spatial and temporal expression and function of TRIM9, one of TRIM family proteins, remain obscure. Our results here showed that TRIM9 protein is mainly expressed in the cerebral cortex, and functions as an E3 ubiquitin ligase collaborating with an E2 ubiquitin conjugating enzyme UbcH5b. Immunohistochemical examination revealed that TRIM9 is localized to the neurons in the normal mouse and human brain and that TRIM9 immunoreactivity is severely decreased in the affected brain areas in Parkinson's disease and dementia with Lewy bodies. This repressed level of TRIM9 protein was supported by immunoblotting analysis. Intriguingly, cortical and brainstem-type Lewy bodies were immunopositive for TRIM9. These results suggest that TRIM9 plays an important role in the regulation of neuronal functions and participates in pathological process of Lewy body disease through its ligase activity.

Dynamic movements of Ro52 cytoplasmic bodies along microtubules.

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren's syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5alpha cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.

Ro52-mediated monoubiquitination of IKK{beta} down-regulates NF-{kappa}B signalling.

Upon activation, NF-kappaB translocates into the nucleus and initiates biological events. This NF-kappaB signalling is mainly regulated by the protein kinase IKKbeta. Early in this signalling pathway, IKKbeta is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of HTLV-1. In cells infected by HTLV-1, IKKbeta is persistently phosphorylated and conjugated with monoubiquitin due to Tax expression. Although this Tax-induced monoubiquitination appears to be an important regulation system for IKKbeta, how the monoubiquitination occurs is unknown and its role in NF-kappaB signalling is still unclear. Here, we show that an E3-ubiquitin ligase Ro52 interacts weakly with wild-type IKKbeta but strongly with a phosphomimetic mutant IKKbeta to conjugate monoubiquitin in cooperation with an E2-ubiquitin-conjugating enzyme UbcH5B. These results suggest that the Tax-induced phosphorylation of IKKbeta causes an interaction with Ro52 for the subsequent monoubiquitination. NF-kappaB reporter assays have shown that the IKKbeta activity is suppressed by wild-type Ro52, but not by its inactive mutant. In addition, monoubiquitin fusion of IKKbeta reduced its activity for NF-kappaB signalling. We also found that Ro52 dramatically reduces the level of Tax. These results suggest that Ro52 down-regulates Tax-induced NF-kappaB signalling by monoubiquitinating IKKbeta and by reducing the level of Tax.