High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization.

Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically recombined with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 µL) incubated for only 72 h. Analysis of eight crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.

Impact of prior bevacizumab therapy on the incidence of ramucirumab-induced proteinuria in colorectal cancer: a multi-institutional cohort study.

BACKGROUND: The association between prior bevacizumab (BEV) therapy and ramucirumab (RAM)-induced proteinuria is not known. We aimed to investigate this association in patients with metastatic colorectal cancer (mCRC). METHODS: mCRC patients who received folinic acid, fluorouracil, and irinotecan (FOLFIRI) plus RAM were divided into with and without prior BEV treatment groups. The cumulative incidence of grade 2-3 proteinuria and rate of RAM discontinuation within 6 months (6M) after RAM initiation were compared between the two groups. RESULTS: We evaluated 245 patients. In the Fine-Gray subdistribution hazard model including prior BEV, age, sex, comorbidities, eGFR, proteinuria ≥ 2 + at baseline, and later line of RAM, prior BEV treatment contributed to proteinuria onset (P < 0.01). A shorter interval between final BEV and initial RAM increased the proteinuria risk; the adjusted odds ratios (95% confidence intervals) for the intervals of < 28 days, 28-55 days, and > 55 days (referring to prior BEV absence) were 2.60 (1.23-5.51), 1.51 (1.01-2.27), and 1.04 (0.76-1.44), respectively. The rate of RAM discontinuation for ≤ 6M due to anti-VEGF toxicities was significantly higher in the prior BEV treatment group compared with that in the no prior BEV treatment group (18% vs. 6%, P = 0.02). Second-line RAM discontinuation for ≤ 6M without progression resulted in shorter overall survival of 132 patients with prior BEV treatment (P < 0.01). CONCLUSION: Sequential FOLFIRI plus RAM after BEV failure, especially within 55 days, may exacerbate proteinuria. Its escalated anti-VEGF toxicity may negatively impact the overall survival.

Associations among metabolic health, weight status, and various physical fitness components in Japanese adolescents: a cross-sectional study (NICE EVIDENCE study- Agano 3).

Aim was to examine associations among metabolic health, weight status, and various physical fitness (PF) components in 1744 Japanese adolescents aged 13-14. Anthropometric measurements and PF tests (20 m shuttle run test [20mSRT], handgrip strength/body mass [HG], standing long jump [SLJ], and sit ups [SU]) were administered. The bottom sex-specific quintile of PF indicated "low fit". Participants were classified as non-overweight (non-OW) or overweight/obese (OW) according to the International Obesity Task Force. Clustered metabolic risk was defined as the sum of Z scores for mean arterial pressure, non-high-density lipoprotein cholesterol, and HbA1c, divided by three, and ≥ 1 SD. Combination of weight status and scores for HG or SU were additively associated with clustered metabolic risk. Compared with the non-OW-moderate-high fit group, the OW-low HG group was 3.05 (95%CI: 1.88-4.97) times more likely to have clustered metabolic risk although risk was not significantly elevated in the OW-moderate-high HG group (1.52 [95%CI: 0.88-2.62]). A similar association was observed between OW and low SU scores but not between OW and low 20mSRT or SLJ scores. Adolescents with OW and moderate-high HG or SU scores had a lower prevalence of an unfavourable metabolic state than those with OW and low HG or SU results.

Increased SLAMF7high monocytes in myelofibrosis patients harboring JAK2V617F provide a therapeutic target of elotuzumab.

Monocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.

[Long-Term Survival of a Patient with Inoperable Gastric Cancer with Distant Lymph Node Metastasis-A Case Report].

A 76-year-old female patient was diagnosed with inoperable gastric cancer with distant lymph node metastasis(cT3N2M1 [LYM], cStage Ⅳ), for which she received S-1 chemotherapy(orally administered on days 1-14 ofa 28-day courses). The patient received a total of4 2 treatment courses. After an initial phase of stable disease due to chemotherapy, she eventually showed progressive disease. S-1 chemotherapy was discontinued. Because ofher social background, she decided against any further chemotherapy. After 1 year, she underwent metallic stent insertion through the gastric cancer, which enabled her to consume food. She is currently alive as of 5 years and 3 months from the date of first diagnosis.

A new microsporidium Percutemincola moriokae gen. nov., sp. nov. from Oscheius tipulae: A novel model of microsporidia-nematode associations.

Here, we describe a new microsporidium Percutemincola moriokae gen. nov., sp. nov., which was discovered in the intestinal and hypodermal cells of a wild strain of the nematode Oscheius tipulae that inhabits in the soil of Morioka, Iwate Prefecture, Japan. The spores of Pe. moriokae had an average size of 1.0 × 3.8 µm and 1.3 × 3.2 µm in the intestine and hypodermis, respectively, and electron microscopy revealed that they exhibited distinguishing features with morphological diversity in the hypodermis. Isolated spores were able to infect a reference strain of O. tipulae (CEW1) through horizontal transmission but not the nematode Caenorhabditis elegans. Upon infection, the spores were first observed in the hypodermis and then in the intestine the following day, suggesting a unique infectious route among nematode-infective microsporidia. Molecular phylogenetic analysis grouped this new species with the recently identified nematode-infective parasites Enteropsectra and Pancytospora forming a monophyletic sister clade to Orthosomella in clade IV, which also includes human pathogens such as Enterocytozoon and Vittaforma. We believe that this newly discovered species and its host could have application as a new model in microsporidia-nematode association studies.

Molecular dissection of Caenorhabditis elegans ATP-binding cassette transporter protein HAF-4 to investigate its subcellular localization and dimerization.

Caenorhabditis elegans HAF-4 and HAF-9 are half-type ATP-binding cassette (ABC) transporter proteins, which are highly homologous to the human peptide transporter protein, transporter associated with antigen processing-like (TAPL, ABCB9). TAPL forms homodimers and localizes to lysosomes, whereas HAF-4 and HAF-9 form heterodimers and localize to intestine-specific non-acidified organelles. Both TAPL and HAF-4/HAF-9 are predicted to have four amino-terminal transmembrane helices [transmembrane domain 0 (TMD0)] additional to the six transmembrane helices that form the canonical core domain of ABC transporters with a cytosolic ABC region. TAPL requires its amino-terminal domain for localization to lysosomes; however, molecular mechanisms underlying HAF-4 and HAF-9 localization to their target organelles had not been elucidated. Here, we demonstrate that the mechanisms underlying HAF-4 localization differ from those underlying TAPL localization. Using transgenic C. elegans expressing mutant HAF-4 proteins labeled with green fluorescent protein, we reveal that the TMD0 of HAF-4 was not sufficient for proper localization of the protein. The mutant HAF-4, which lacked TMD0, localized to intracellular organelles similarly to the wild-type protein and functioned normally in the biogenesis of its localizing organelles, indicating that the TMD0 of HAF-4 is dispensable for both its localization and function.

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.

BACKGROUND: The intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9). RESULTS: Using transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis. CONCLUSIONS: Our results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.

Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species.

Allergenic characteristics of purified parvalbumins from different fish species have not been thoroughly investigated. We revealed that purified parvalbumins from nine different fish species have identical IgE-reactivities and high cross-reactivities. We also showed that fish allergenicity is associated with the parvalbumin content of the fish species, rather than species-specific differences in the molecular characteristics of the individual parvalbumin proteins.

Spinal cord stimulation modulates supraspinal centers of the descending antinociceptive system in rats with unilateral spinal nerve injury.

BACKGROUND: The descending antinociceptive system (DAS) is thought to play crucial roles in the antinociceptive effect of spinal cord stimulation (SCS), especially through its serotonergic pathway. The nucleus raphe magnus (NRM) in the rostral ventromedial medulla is a major source of serotonin [5-hydroxytryptamine (5-HT)] to the DAS, but the role of the dorsal raphe nucleus (DRN) in the ventral periaqueductal gray matter is still unclear. Moreover, the influence of the noradrenergic pathway is largely unknown. In this study, we evaluated the involvement of these serotonergic and noradrenergic pathways in SCS-induced antinociception by behavioral analysis of spinal nerve-ligated (SNL) rats. We also investigated immunohistochemical changes in the DRN and locus coeruleus (LC), regarded as the adrenergic center of the DAS, and expression changes of synthetic enzymes of 5-HT [tryptophan hydroxylase (TPH)] and norepinephrine [dopamine ß-hydroxylase (DßH)] in the spinal dorsal horn. RESULTS: Intrathecally administered methysergide, a 5-HT1- and 5-HT2-receptor antagonist, and idazoxan, an α2-adrenergic receptor antagonist, equally abolished the antinociceptive effect of SCS. The numbers of TPH-positive serotonergic and phosphorylated cyclic AMP response element binding protein (pCREB)-positive neurons and percentage of pCREB-positive serotonergic neurons in the DRN significantly increased after 3-h SCS. Further, the ipsilateral-to-contralateral immunoreactivity ratio of DßH increased in the LC of SNL rats and reached the level seen in naïve rats, even though the number of pCREB-positive neurons in the LC was unchanged by SNL and SCS. Moreover, 3-h SCS did not increase the expression levels of TPH and DßH in the spinal dorsal horn. CONCLUSIONS: The serotonergic and noradrenergic pathways of the DAS are involved in the antinociceptive effect of SCS, but activation of the DRN might primarily be responsible for this effect, and the LC may have a smaller contribution. SCS does not potentiate the synthetic enzymes of 5HT and norepinephrine in the neuropathic spinal cord.

Amino-terminal extension of 146 residues of L-type GATA-6 is required for transcriptional activation but not for self-association.

Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association.

Prednisone versus high-dose dexamethasone for untreated primary immune thrombocytopenia. A retrospective study of the Japan Hematology & Oncology Clinical Study Group.

High-dose dexamethasone (HDD) has been shown to be an effective initial treatment for immune thrombocytopenia (ITP), but it is not clear whether HDD offers any advantages over conventional-dose prednisone (PSL). We retrospectively compared the efficacy and toxicity of HDD and PSL for newly diagnosed ITP. The response was evaluated according to the International Working Group (IWG) criteria. We analyzed data from 31 and 69 patients in the HDD and PSL groups, respectively. There were no significant differences in patient characteristics between the two groups except for the incidence of the eradication of Helicobacter pylori. The response rate was better in the HDD group (42.7 vs. 28.4 %), and this difference was statistically significant when adjusted for other factors including the eradication of H. pylori. In the HDD group, a response was achieved earlier (28 vs. 152 days in median) and steroids were more frequently discontinued at 6 months (64.5 vs. 37.7 %). Among patients who achieved a response, there was no significant difference in the incidence of loss of response. There were no significant differences in the rate of adverse events, transition to chronic ITP, and splenectomy. In conclusion, HDD might enable the early cessation of steroids without a loss of response.

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans.

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Cross-Sectional and Longitudinal Associations between Forearm Bone Mineral Density and Anthropometry in Adult Japanese Men and Women.

BACKGROUND: No consensus exists regarding which anthropometric measurements are related to bone mineral density (BMD), and this relationship may vary according to sex and age. A large Japanese cohort was analyzed to provide an understanding of the relationship between BMD and anthropometry while adjusting for known confounding factors. METHODS: Our cohort included 10,827 participants who underwent multiple medical checkups including distal forearm BMD scans. Participants were stratified into four groups according to age (≥50 years or <50 years) and sex. The BMD values were adjusted for confounding factors, after which single and partial correlation analyses were performed. The prevalence of osteopenia was plotted for each weight index (weight or body mass index [BMI]) class. RESULTS: Cross-sectional studies revealed that weight was more favorably correlated than BMI in the older group (R=0.278 and 0.212 in men and R=0.304 and 0.220 in women, respectively), whereas weight and BMI were weakly correlated in the younger age groups. The prevalence of osteopenia exhibited a negative linear relationship with weight among older women ≥50 years of age, and an accelerated increase was observed with decreasing weight in older men weighing <50 kg and younger women weighing <60 kg. When weight was replaced with BMI, the prevalence was low in most subgroups classified by weight. CONCLUSIONS: Weight, rather than BMI, was the most important indicator of osteopenia but it might not be predictive of future bone loss.

Retrospective assessment of secondary prophylaxis for invasive aspergillosis in neutropenic hematology patients and identification of risk factors for relapse of fungal disease.

BACKGROUND: Invasive aspergillosis (IA) is a critical complication in neutropenic patients. Recurrent IA is especially associated with high mortality. Therefore, secondary prophylaxis is important in patients with a history of IA. We retrospectively assessed the effect of secondary prophylaxis for IA. METHODS: We reviewed the medical records of 46 hematology patients who developed possible, probable, or proven IA according to the EORTC/MSG criteria between 2005 and 2009, and who subsequently underwent chemotherapy (n = 30) or stem cell transplantation (n = 16). RESULTS: Ten patients developed recurrent IA within 10 days after recovery from neutropenia. None of the 15 patients who achieved complete response (CR) of IA experienced recurrent IA. Among patients who did not achieve CR of IA, multivariate analysis identified the following independent risk factors: female sex (hazard ratio (HR) 7.23, 95% confidence interval (CI) 2.38-21.9, p = 0.00047), high serum C-reactive protein level (≥ 1 mg/dl) at the beginning of subsequent therapy (HR 4.46, 95% CI 1.51-13.2, p = 0.007), and the use of micafungin (HR 12.0, 95% CI 2.03-71.2, p = 0.0061) or amphotericin B (HR 16.5, 95% CI 1.56-174, p = 0.020) for secondary prophylaxis (reference: voriconazole). CONCLUSIONS: Three risk factors for recurrent IA were identified. However, a prospective controlled trial is required to evaluate the impact of secondary prophylactic regimens.

Baseline and seasonal trends of Bacillus cereus and Bacillus subtilis from clinical samples in Japan.

Background: Outbreaks of Bacillus cereus bloodstream infections (BSIs) are a concern in Japanese medical settings. Aim: This study determined baseline values for B. cereus detection in clinical samples that are useful as reference values for hospitals when assessing the need for intervention. Method: A retrospective analysis of B. cereus detection in the Japan Nosocomial Infections Surveillance data from 2008 to 2014 was performed; it included 950 individual hospitals across the country. Findings: Bacillus spp. were detected in 0.54% of the clinical specimens submitted for bacteriological testing. Specimens positive for Bacillus spp. were mainly blood (24.6%), stool (26.5%), and respiratory specimens (23.3%). Identification of Bacillus spp. at the species level (i.e., B. cereus or B. subtilis) was reported in 55.3%, 14.7%, and 15.4% of cases, of which 88.9%, 48.3%, and 33.1% were B. cereus in blood, stool, and respiratory specimens, respectively. Of the 4105 hospital-years, 75.7% had blood specimens with Bacillus spp., with a median of 0.85 blood specimens/100 beds annually (interquartile range, 0.17-2.10). The B. cereus detection showed significant summer seasonality, regardless of specimen type or geographic distribution. The B. subtilis detection did not show seasonality, and its detection remained constant throughout the year. The seasonality of Bacillus spp. reflects the high proportion of B. cereus. Conclusions: The increased detection rate of Bacillus spp. during summer should be interpreted as a risk factor for B. cereus BSIs. A post-summer decrease in Bacillus spp. should not be interpreted as an effect of interventions.

Characteristics, Outcomes, and Risk Factors for Upper Gastrointestinal Bleeding in Inpatients - A Comparison with Outpatients.

Objective The study objectives were to clarify the clinical findings and the causes of intractability and mortality of upper gastrointestinal (UGI) bleeding in inpatients. Methods The patients were divided into Inpatient (Ip) and Outpatient (Op) onset groups, and their characteristics, clinical and bleeding data, and outcomes were compared. Patients Our study included 375 patients who developed UGI bleeding during hospitalization or were admitted after being diagnosed with UGI bleeding in an outpatient setting from January 1, 2015, to June 30, 2020. Results The Ip group had worse general condition; increased percentages of comorbidities; and more common use of proton pump inhibitor, anti-coagulant, and steroid than the Op group. Compared with the Op group, the Ip group had lower serum albumin levels and platelet counts at the onset of bleeding, whereas rebleeding, mortality, and bleeding-related death rates were higher. Multivariate analysis of the Ip group revealed that the risks of rebleeding included endoscopic high-risk stigmata, maintenance dialysis, and duodenal bleeding, whereas the risks of mortality were gastric ulcer and a Charlson Comorbidity Index update score of ≥3. Conclusion UGI bleeding in the Ip group was associated with higher rebleeding and mortality rates. Because of their poor general health condition, the pathology of UGI bleeding in these patients may differ from that of patients with common UGI bleeding. A different approach for the care and prevention of UGI bleeding in inpatients is required.

Combined effects of blood pressure and glycemic status on risk of heart failure: a population-based study.

AIMS: To investigate the combined effects of blood pressure (BP) and glycemic status on the risk of heart failure. METHODS: Examined was a Japanese claims database from 2008 to 2019 on 589 621 individuals. Cox proportional hazards model identified the incidence of heart failure among five levels of SBP/DBP according to glucose status. RESULTS: Mean follow-up period was 5.6 years. The incidence of heart failure per 1000 person-years in the normoglycemia, borderline glycemia, and diabetes groups were 0.10, 0.18, and 0.80, respectively. In normoglycemia, a linear trend was observed between both SBP and DBP categories and hazard ratios for heart failure ( P for linearity <0.001). In borderline glycemia, J-shaped association was observed between DBP categories and hazard ratios, although the liner trend was significant ( P  < 0.001). In diabetes, the linear trend for the relationship between DBP categories and hazard ratios was not significant ( P  = 0.09) and the J-shaped association in relation to the hazard ratios was observed between SBP categories and heart failure risk. In the lowest SBP category (i.e. SBP < 120 mmHg), patients with diabetes had more than five-fold heart failure risk [hazard ratio (95% confidence interval), 5.10 (3.19-8.15)], compared with those with normoglycemia and SBP less than 120 mmHg. CONCLUSION: The association between SBP/DBP and heart failure risk weakened with worsening of glucose metabolism, suggesting strict BP control accompanied by excessively lowered DBP should be cautious in prevent heart failure in abnormal glycemic status. Particularly in diabetes, comprehensive management of risk factors other than BP may be essential to prevent heart failure. Further trials are needed to support these suggestions and apply them to clinical practice.

Cell-free protein crystallization for nanocrystal structure determination.

In-cell protein crystallization (ICPC) has been investigated as a technique to support the advancement of structural biology because it does not require protein purification and a complicated crystallization process. However, only a few protein structures have been reported because these crystals formed incidentally in living cells and are insufficient in size and quality for structure analysis. Here, we have developed a cell-free protein crystallization (CFPC) method, which involves direct protein crystallization using cell-free protein synthesis. We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 µL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined. We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.

Metalloproteinase-Dependent and TMPRSS2-Independent Cell Surface Entry Pathway of SARS-CoV-2 Requires the Furin Cleavage Site and the S2 Domain of Spike Protein.

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. IMPORTANCE To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.