Adult T-cell leukemia/lymphoma complicated by Pneumocystis pneumonia in a non-endemic area.

Adult T-cell leukemia/lymphoma (ATLL) is a human T-cell leukemia virus type 1-inducing unevenly-distributed T-cell malignancy, which is often complicated by opportunistic infections. Here, we discuss the case of a 75-year-old woman presenting with Pneumocystis pneumonia (PCP) who was subsequently diagnosed with ATLL in Tokyo, a non-endemic area of ATLL. In addition to the elevated soluble interleukin-2 receptor and the detection of flower cells in the screening blood test, the high-resolution computed tomography findings, atypical of PCP, were clues to the diagnosis of ATLL. ATLL should be considered as an underlying disease when patients present with PCP, even in non-endemic areas.

Clinical Utility of Rapid On-Site Evaluation of Touch Imprint Cytology during Cryobiopsy for Peripheral Pulmonary Lesions.

Cryobiopsy enables us to obtain larger specimens than conventional forceps biopsy despite the caution regarding complications. This study aimed to evaluate the clinical utility of rapid on-site evaluation of touch imprint cytology (ROSE-TIC) during cryobiopsy of peripheral pulmonary lesions (PPLs). We retrospectively reviewed the data of consecutive patients who underwent cryobiopsy for solid PPLs between June 2020 and December 2021. ROSE-TIC was performed on the first specimen obtained via cryobiopsy and assessed using Diff-Quik staining. The results of ROSE-TIC for each patient were compared with the histological findings of the first cryobiopsy specimen. Sixty-three patients were enrolled in this study. Overall, 57 (90.5%) lesions were ≤30 mm in size and 37 (58.7%) had positive bronchus signs. The radial endobronchial ultrasound findings were located within and adjacent to the lesion in 46.0% and 54.0% of the cases, respectively. The sensitivity, specificity, and positive and negative predictive values of the ROSE results for histological findings of the corresponding specimens were 69.8%, 90.0%, 93.8%, and 58.1%, respectively. The concordance rate was 76.2%. In conclusion, ROSE-TIC, due to its high specificity and positive predictive value, may be a potential tool in deciding whether cryobiopsy sampling could be finished during bronchoscopy.

Unilateral Autoimmune Pulmonary Alveolar Proteinosis with Polymyositis-related Interstitial Lung Disease.

A 61-year-old patient with cystic bronchiectasis and bronchial artery hyperplasia in the left lung was diagnosed with polymyositis-related interstitial lung disease. After nine months of immunosuppressive therapy, he developed unilateral autoimmune pulmonary alveolar proteinosis (APAP) in the right lung with respiratory failure. After bronchial artery embolization to prevent massive hemoptysis, whole-lung lavage was performed using veno-venous extracorporeal membrane oxygenation. His respiratory condition improved, and he was discharged from the hospital with supplemental oxygen. Three reported cases of APAP with polymyositis-related interstitial lung disease, including the present case, were all positive for anti-glycyl tRNA synthetase antibody and were under immunosuppressive treatment.

Clinical characteristics of pulmonary hypertension in patients with pleuroparenchymal fibroelastosis.

BACKGROUND: This study aimed to investigate the clinical characteristics and prognosis of patients with pleuroparenchymal fibroelastosis (PPFE) and pulmonary hypertension (PH). METHODS: We retrospectively analyzed the data of patients who were diagnosed with PPFE and underwent transthoracic echocardiography (TTE) for the evaluation of their right heart systems within 3 months of their first visit between 2011 and 2018. Patients were divided into the PH and non-PH groups based on their peak tricuspid regurgitation velocity (TRV) on TTE (cutoff, 2.8 m/s). The clinical characteristics of PH and association between PH and survival among patients with PPFE were investigated. RESULTS: In total, 83 patients were enrolled. Sixteen (19.3%) patients were included in the PH group. The PH group had a lower body mass index, percent predicted forced vital capacity (FVC), 6-min walk distance, and partial pressure of arterial oxygen than the non-PH group. There was no significant difference in the presence of usual interstitial pneumonia patterns in the lower lobes between the two groups. The survival period was significantly shorter in the PH group than in the non-PH group (median survival 16.3 versus 50.2 months, log-rank p < 0.001). The multivariate Cox proportional hazard model showed that male sex (hazard ratio [HR] = 4.83, p < 0.001), Krebs von den Lungen-6 (KL-6) > 550 U/mL (HR = 3.48, p = 0.005), %FVC < 50% (HR = 3.04, p = 0.028), and peak TRV > 2.8 m/s (HR = 3.26, p = 0.038) were independently associated with poor survival. CONCLUSIONS: PH was not rare in patients with PPFE. Male sex, increased KL-6, lower FVC, and PH were independently associated with poor survival in patients with PPFE.

Therapeutic options in thymomas and thymic carcinomas.

INTRODUCTION: Curative-intent surgery is the best therapeutic option for thymic malignancies. However, patients with advanced or recurrent thymic malignancies often require palliative-intent chemotherapy or radiotherapy. Since thymic malignancies are rare cancers, the efficacy and safety of treatments have been verified based on small Phase 2 trials or retrospective studies. AREA COVERED: We comprehensively reviewed the treatment strategies for thymic malignancies, including surgery, radiotherapy, and pharmacotherapy, including cytotoxic chemotherapy, molecular-targeted therapy, and immunotherapy. Additionally, we reviewed specific situations, such as pleural dissemination, central nervous system metastasis, and paraneoplastic syndrome. EXPERT OPINION: Cytotoxic chemotherapy remains the standard option in pharmacotherapy. However, multikinase inhibitors, such as sunitinib and lenvatinib, and immune checkpoint inhibitors including pembrolizumab have been developed to treat thymic carcinomas. Now, a Phase 2 study is evaluating whether lenvatinib plus pembrolizumab benefits patients with type B3 thymoma or thymic carcinoma. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin inhibitors may contribute to disease control and octreotide scan is only applicable to somatostatin analogues. Although the genomic characteristics of thymic malignancies have been analyzed, few actionable mutations have been detected in general. The development of a treatment strategy using combination pharmacotherapy is anticipated.

An autopsy case of idiopathic pulmonary fibrosis with remarkable honeycomb cyst expansion.

Herein, we report an autopsy case of idiopathic pulmonary fibrosis (IPF) in which remarkable honeycomb cyst expansion appeared in the clinical course. Radiological findings initially showed subpleural predominant reticulation that had progressed to usual interstitial pneumonia with honeycomb cysts, along with a restrictive pattern in the pulmonary function tests. The diameter of honeycomb cysts had gradually increased, and some cysts had abruptly expanded at the end stage. Based on pathological findings of autopsy specimens, bronchiectasis, alveolar collapse due to inflammation, and check-valve mechanism caused by a slit-like orifice of the cysts could have contributed to honeycomb cyst expansion.

Drug-induced hypersensitivity syndrome caused by minodronic acid hydrate.

BACKGROUND: Drug-induced hypersensitivity syndrome (DIHS)/drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is an important adverse reaction caused by a few drugs. Reactivation of human herpesvirus 6 (HHV-6) is known to be associated with its pathogenesis. DIHS occasionally manifests as pulmonary lesions with a variety of imaging findings. CASE PRESENTATION: An 83-year-old woman started taking minodronic acid hydrate 5 years before admission. She noticed a generalized skin rash 44 days before admission and started oral betamethasone-d-chlorpheniramine maleate combination tablets for allergic dermatitis. She developed a fever and cough in addition to the rash, and was referred to our hospital. Laboratory data showed a high level of eosinophils and liver and biliary enzymes. Computed tomography (CT) studies revealed bilateral diffuse ground-glass opacities with ill-defined centrilobular nodules from the central to peripheral regions of the lungs. Transbronchial lung cryobiopsy specimens showed that lymphocyte infiltration was observed in the alveolar walls and fibrinous exudates and floating macrophages in the alveolar lumina. Immunohistochemistry of biopsy specimens showed more CD4+ lymphocytes than CD8+ lymphocytes, while few Foxp3+ lymphocytes were recognized. The serum anti-HHV-6 immunoglobulin G titer increased at 3 weeks after the first test. Based on these findings, we diagnosed her with DIHS. We continued care without using corticosteroids since there was no worsening of breathing or skin condition. Eventually, her clinical symptoms chest CT had improved. Minodronic acid hydrate was identified as the culprit drug based on the positive results of the patch test and drug-induced lymphocyte stimulation test. CONCLUSIONS: We described the first case of DIHS caused by minodronic acid hydrate. Lung lesions in DIHS can present with bilateral diffuse ground-glass opacities and ill-defined centrilobular nodules on a CT scan during the recovery phase. Clinicians should be aware of DIHS, even if patients are not involved with typical DIHS/DRESS-causing drugs.

A case of pulmonary sclerosing pneumocytoma diagnosed preoperatively using transbronchial cryobiopsy.

BACKGROUND: The preoperative diagnosis of pulmonary sclerosing pneumocytoma (PSP) is complicated since PSP has several histological structural patterns in the same neoplasm; hence, it is sometimes pathologically misdiagnosed as adenocarcinoma or carcinoid. In recent years, with the prevalence of transbronchial cryobiopsy (TBLC), we are able to obtain larger specimens than previously. However, to date, there have been no reports describing PSP diagnosed using TBLC. CASE REPORTS: A 43-year-old man was referred to our hospital for an abnormal lesion in the left lung discovered on routine health examination. A computed tomography scan of the chest revealed a 14-mm heterogeneous round nodule with surrounding ground-glass opacity in the left lower lobe. The tumor size increased to 18 mm in three weeks, and he developed bloody sputum. TBLC was performed using radial endobronchial ultrasonography and fluoroscopy. An occlusion balloon and prophylactic epinephrine were used to prevent severe bleeding. Histologically, epithelioid cells with solid proliferation, various papillary lesions, and hemosiderin-laden histiocytes were observed. Immunohistochemical staining revealed the histiocytes positive for thyroid transcription factor-1 and vimentin, and the type II pneumocyte-like-cells positive for cytokeratin 7. The tumor was preoperatively diagnosed as a PSP; the patient underwent left basal segmentectomy and consequently, a final diagnosed of PSP was formulated. CONCLUSION: We report the first case of PSP preoperatively diagnosed using TBLC. Therefore, cryobiopsy could be beneficial in the preoperative diagnosis of PSP.

Clinical impact of combination therapy with baricitinib, remdesivir, and dexamethasone in patients with severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has spread worldwide and is also an important disease in Japan. Thus, the optimal treatment strategy for severe COVID-19 should be established urgently. The effects of combination treatment with baricitinib-a Janus kinase inhibitor, remdesivir, and dexamethasone (BRD) are unknown. METHODS: Patients who received combination therapy with BRD at the Japanese Red Cross Medical Center were enrolled in the study. All patients received baricitinib (≤14 d), remdesivir (≤10 d), and dexamethasone (≤10 d). The efficacy and adverse events were evaluated. RESULTS: In total, 44 patients with severe COVID-19 were enrolled in this study. The 28-d mortality rate was low at 2.3% (1/44 patients). The need for invasive mechanical ventilation was avoided in most patients (90%, 17/19 patients). Patients who received BRD therapy had a median hospitalization duration of 11 d, time to recovery of 9 d, duration of intensive care unit stay of 6 d, duration of invasive mechanical ventilation of 5 d, and duration of supplemental oxygen therapy of 5 d. Adverse events occurred in 15 patients (34%). Liver dysfunction, thrombosis, iliopsoas hematoma, renal dysfunction, ventilator-associated pneumonia, infective endocarditis, and herpes zoster occurred in 11%, 11%, 2%, 2%, 2%, 2%, and 2% of patients, respectively. CONCLUSIONS: Combination therapy with BRD was effective in treating severe COVID-19, and the incidence rate of adverse events was low. The results of the present study are encouraging; however, further randomized clinical studies are needed.

Medical costs of Japanese lung cancer patients during end-of-life care.

OBJECTIVE: The medical costs associated with cancer treatment have increased rapidly in Japan; however, little data exist on actual costs, especially for end-of-life care. Therefore, this study aimed to examine the medical costs of lung cancer patients during the last 3 months before death and to compare the costs with those of initial anticancer treatment. METHODS: We retrospectively evaluated all patients who died from lung cancer at the Japanese Red Cross Medical Center between 1 January 2008 and 31 August 2019. Patients were classified into three cohorts (2008-2011, 2012-2015 and 2016-2019) according to the year of death; the medical costs were evaluated for each cohort. Costs were then divided into outpatient and inpatient costs and calculated per month. RESULTS: Seventy-nine small cell lung cancer and 213 non-small cell lung cancer patients were included. For small cell lung cancer and non-small cell lung cancer patients, most end-of-life medical costs were inpatient costs across all cohorts. The median monthly medical costs for the last 3 months among both small cell lung cancer and non-small cell lung cancer patients did not differ significantly among the cohorts, but the mean monthly costs for non-small cell lung cancer tended to increase. The monthly medical costs for the last 3 months were significantly higher than those for the first year in SCLC (P = 0.013) and non-small cell lung cancer (P < 0.001) patients and those for the first 3 months in non-small cell lung cancer patients (P = 0.005). CONCLUSIONS: The medical costs during the end-of-life period for lung cancer were high and surpassed those for initial treatment.

Medical costs of lung cancer care in Japan during the first one or two years after initial diagnosis.

OBJECTIVES: Japan's healthcare expenditures, especially on oncology, are rapidly growing; however, there are scant data on actual costs and cost-effectiveness in the real world. The aim was to assess the medical costs and outcomes of patients with advanced lung cancer. METHODS: We retrospectively investigated all patients who were diagnosed with advanced lung cancer at the Japanese Red Cross Medical Center between 1 January 2008 and 31 December 2018. Patients were classified into three cohorts according to the year of diagnosis-Cohort 1: 2008-2010, Cohort 2: 2011-2014 and Cohort 3: 2015-2018-and assessed for medical costs and outcome. Medical costs were divided into outpatient and inpatient costs and were calculated on a monthly basis. RESULTS: Ninety-five patients with small cell lung cancer (SCLC) and 330 with nonsmall cell lung cancer (NSCLC) were included. There was a trend toward increased costs during the first two years after diagnosis in NSCLC patients, without changes in monthly costs, reflecting improved survival. Compared to Cohort 1, Cohort 3 patients with NSCLC had longer survival (median: 24 versus 12 months, P < 0.001), with a median incremental cost of Japanese Yen 6 million during the initial two years. The proportion of outpatient costs increased over time, especially for NSCLC patients (P < 0.001). No changes in costs or survival were observed in SCLC patients. CONCLUSIONS: In NSCLC patients, medical costs increased with prolonged survival during the last decade. The costs on a monthly basis did not change. The proportion of outpatient costs increased.

Utility of radial endobronchial ultrasonography combined with transbronchial lung cryobiopsy in patients with diffuse parenchymal lung diseases: a multicentre prospective study.

BACKGROUND: Radial endobronchial ultrasonography (R-EBUS) has been used in conjunction with transbronchial lung cryobiopsy (TBLC) to diagnose diffuse parenchymal lung disease (DPLD) and to decrease the risk of bleeding complications. The diagnostic utility of different R-EBUS signs, however, remains unknown. OBJECTIVES: This study aimed to determine whether different R-EBUS signs could be used to more accurately diagnose DPLD and whether bronchial bleeding could be prevented with use of R-EBUS during TBLC. METHOD: Eighty-seven patients with DPLD were included in this multicentre prospective study, with 49 patients undergoing R-EBUS. R-EBUS signals were characterised as displaying either dense or blizzard signs. Pathological confidence of specimens obtained from TBLC was compared between patients with dense versus blizzard signs, and severity of bronchial bleeding was determined based on whether R-EBUS was performed or not. RESULTS: All patients with dense signs on R-EBUS showed consolidation on high-resolution CT (HRCT) imaging. Pathological confidence of lung specimens was significantly higher in patients with dense signs versus those with blizzard signs (p<0.01) and versus those who did not undergo R-EBUS (p<0.05). Patients who underwent TBLC with R-EBUS were more likely to experience no or mild bronchial bleeding than patients who did not undergo R-EBUS (p<0.01), with shorter procedure times (p<0.01). CONCLUSIONS: The dense R-EBUS sign corresponded with consolidation on HRCT. High-quality lung specimens may be obtainable when the dense sign is observed on R-EBUS, and R-EBUS combined with TBLC may reduce risk of bronchial bleeding and shorten procedure times.

1H, 13C and 15N resonance assignment of the YTH domain of YTHDC2.

In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m6A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m6A differs between these proteins. Here, we report 1H, 13C and 15N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a ß1-α1-ß2-α2-ß3-ß4-ß5-α3-ß6-α4 topology, which was also observed in YTH1. However, the ß4-ß5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m6A-binding pocket of YTH2 formed by the ß4-ß5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.

Comparison of adequacy between transbronchial lung cryobiopsy samples and endobronchial ultrasound-guided transbronchial needle aspiration samples for next-generation sequencing analysis.

BACKGROUND: Most lung cancer patients present with lesions in both lung fields and lymphadenopathy. Thus, transbronchial lung cryobiopsy (TBLC) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are commonly performed for diagnosing lung cancer. However, the adequacy of these samples for next-generation sequencing (NGS) analysis remains unclear. This study aimed to compare the adequacy between TBLC and EBUS-TBNA samples for NGS analysis. METHODS: This retrospective cohort study included patients whose lung samples were collected via TBLC or EBUS-TBNA and analyzed using NGS. Out of 46 genes, the number of genes in TBNA and TBLC samples that could not be assessed via NGS analysis was mainly evaluated. RESULTS: A total of 37 patients were included and classified into two groups (TBLC group, n = 18 and TBNA group, n = 19). The mean number of genes that could not be evaluated via NGS analysis was significantly lower in the TBLC group than in the TBNA group (0.9 vs. 10.3, P = 0.024). The median total area of tumor cells in TBLC samples was significantly greater than that in TBNA samples (6.3 [1.6-4.2] vs. 2.6 [0.2-17.3] mm2 , P < 0.01). In the TBNA group, there were two fully inadequate samples for NGS analysis with a high degree of cell crush or low tumor content, while there was no fully inadequate sample in the TBLC group. CONCLUSIONS: TBLC is more effective in obtaining adequate samples for NGS analysis than EBUS-TBNA. TBLC should be performed to obtain adequate samples for NGS analysis in lung cancer patients with target lesions in lung fields, even if they have lymphadenopathy. KEY POINTS: Significant findings of the study The mean number of genes that could not be evaluated was significantly lower in TBLC samples than in EBUS-TBNA samples (0.9 vs. 10.3, P = 0.024). TBLC could obtain adequate samples with a high concentration of uncrushed tumor cells for NGS. What this study adds To obtain samples for NGS analysis, the use of TBLC should be aggressively considered in lung-cancer patients with target lesions located in lung fields, even if they have lymphadenopathy.

Elucidation of the aberrant 3' splice site selection by cancer-associated mutations on the U2AF1.

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

Clinicopathological and molecular analysis of SIRT7 in hepatocellular carcinoma.

Sirtuin 7 (SIRT7) is a NAD+ (nicotinamide adenine dinucleotide) dependent deacetylase that is reported to contribute to tumour growth and invasion by selectively acting on histone H3K18. It is overexpressed in several cancers including hepatocellular carcinoma (HCC). In this study, we investigated the relationship between SIRT7 expression, proliferation (Ki-67 index) in human HCC tissues, and patient prognosis. We analysed 219 HCC samples obtained retrospectively, for clinicopathological features, and with immunohistochemistry. SIRT7 overexpression was observed in 73 cases (33%) and correlated with vascular invasion and poor differentiation of HCC. Ki-67 labelling index was observed to be significantly higher in SIRT7 overexpressing cases. Interestingly, the Ki-67 labelling index was higher in SIRT7 overexpressing cases regardless of the differentiation status of HCC. Multivariate analysis demonstrated SIRT7 overexpression as an independent factor predictive of poor prognosis (p=0.016). In vitro, SIRT7 knockdown led to reduced growth in cells and resulted in a lower percentage of G0/G1 cells compared to controls. In addition, the ratio of apoptotic cells following sorafenib treatment was significantly higher in SIRT7 knockdown cells than control cells (p=0.040), implying that SIRT7 knockdown potentiated the effect of sorafenib. In conclusion, our study showed that overexpression of SIRT7 was associated with increased proliferative activity in HCC and predictive of poor prognosis. In addition, our in vitro model showed that SIRT7 knockdown was associated with reduced proliferation, and suggested abrogation of SIRT7 may potentiate the effect of sorafenib. Therefore, we propose that SIRT7 expression by HCC may be used as a prognostic biomarker, and that SIRT7 may be a potential target for new therapeutic modalities.

A novel 3' splice site recognition by the two zinc fingers in the U2AF small subunit.

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the ß sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

Glycolytic flux controls D-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes.

D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with D-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of D-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls D-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.

Aberrant assembly of RNA recognition motif 1 links to pathogenic conversion of TAR DNA-binding protein of 43 kDa (TDP-43).

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant ß-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways.

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.