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Background Liver cancer is one of the most common malignancies in the world with a poor prognosis. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, accounting for 80–90% of cases. The initiation and progression of HCC are closely associated with chronic liver inflammation. In addition, HCC is often accompanied by cell senescence. Senescent hepatocytes can secrete various inflammatory factors, collectively called the senescence-associated secretory phenotype (SASP). The SASP has been confirmed to promote the occurrence of liver cancer by affecting the inflammatory microenvironment. However, its role and the underlying mechanism of hepatic SASP in hepatocarcinogenesis are not clearly understood. Therefore, a better understanding of the pathogenic mechanisms of the effect of the hepatic SASP on the occurrence of HCC is still needed. Methods The study aims to explore the role of SASP factors and the underlying mechanism in tumorigenesis and the progression of HCC in vivo. We used diethylnitrosamine (DEN) combined with carbon tetrachloride (CCl4) (DEN-CCl4) to establish liver cancer model in wild-type (WT) mice and Bcl3 knockout (Bcl3−/−) mice. β-galactosidase (β-gal) staining was performed to evaluate the degree of cellular senescence. Immunohistochemistry (IHC) were used to detect the degree of cellular senescence and the activation of macrophage. PCR chip and clinical tissue chip assays were used to estimate the RNA levels of SASP factors and NF-κB related genes, and their protein levels were examined by Western blot assays. Results DEN-CCl4 induced cellular senescence in mouse hepatocytes. In addition, senescent hepatocytes might release a variety of inflammatory factors that further activate macrophages, thereby changing the microenvironmental state and promoting the occurrence of HCC. Mechanistically, the NF-κB pathway is important because it regulates the SASP. Therefore, we used a PCR chip to detect the expression of NF-κB-related genes in senescent liver tissue. Our results showed that the expression of Bcl3 was increased in senescent hepatocytes, and knocking out Bcl3 significantly inhibited the secretion of hepatocyte SASP factors and the activation of macrophages, thereby inhibiting hepatocarcinogenesis. Finally, in clinical tissues adjacent to HCC tissues in patients, the expression of Bcl3 and IL-8 correlated with poor prognosis in HCC patients. Conclusion The hepatic SASP can further induce the activation of macrophages during hepatocarcinogenesis, thereby promoting the occurrence of HCC, and that this process is closely related to the expression of Bcl3 in hepatocytes.

ObjectiveAutoimmune encephalitis (AE) is a large category disorder urging antibody characterization. The aim was to identify a novel AE related autoantibody targeting an intracellular synaptic protein.MethodsSuspected AE patients had negative conventional antibodies screening but strong immunolabel signals on rat brain sections with the serum and cerebrospinal fluid (CSF) samples were considered burdening unkown antibody. Immunoprecipitation from the rat brain protein lysate followed by mass spectrometry analysis was used to identify the targeting antigen. Western blotting and/or cell-based assay (CBA) with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope and IgG subtype determination. Patients with similar immunostaining pattern on rat brain sections were retrospectively screened for the antibody.ResultsThe antibody against collapsin response mediator protein 2 (CRMP2), a synaptic protein involved in axon guidance, was identified in a patient with suspected AE. The patient samples reactivated with HEK293T cell overexpressed CRMP2, rather than CRMP1, 3, 4, and 5. The patient samples mainly stained neuronal cytoplasm of the cortex, hippocampus and cerebellum Purkinje cells. This reactivity was eliminated by pre-immunoabsorption with CRMP2-overexpressing HEK293T cells. CRMP2 truncation experiments indicated that 536 amino acids at C-terminus was necessary for the epitope. Subtype analysis showed that anti-CRMP2 antibody was IgG4. Moreover, screening from 19 suspected AE patients led to identification of anti-CRMP2 antibody in another patient with a diagnosis of encephalomyelitis. The two patients responded to immunotherapy.ConclusionsThis study discovered a novel anti-CRMP2 antibody associated with AE. Testing of the antibody might be promising for AE diagnosis and treatment.

Immune checkpoint inhibitors (ICIs) have come to play an increasingly prominent role in the treatment of lung cancer, and some are recommended as a first-line treatment for late-stage non-small-cell lung cancer, either as a monotherapy or in combination with chemotherapy. Accordingly, the indications of Food and Drug Administration-approved ICIs have increased. In this background, China has implemented various policies to encourage and accelerate the marketing of domestic and imported innovative antitumor drugs. Eight ICIs have been approved in China. Among these, four imported programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have received approval for six indications, and one domestic PD-1 inhibitor has received approval for one indication for lung cancer in 2018. Numerous clinical trials of ICIs for lung cancer are underway in China. This review aims to summarize the recent advances and future directions of ICIs, including PD-1 inhibitors, PD-L1 inhibitors, cytotoxic T lymphocyte-associated antigen-4 inhibitors, bi-specific antibodies, and a novel inhibitor of T-cell immune-receptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains in immunotherapies for lung cancer in China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the Spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures to contain the spread of the disease in many countries has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread and whether they can cause a more severe disease remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (KD) values using surface plasmon resonance (SPR) assays of three fastspreading SARS-CoV-2 variants, Alpha, Beta and Gamma ones, as well as genetic factors in the host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased Spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN and TMPRSS2), may have few contributions to the SARS-CoV-2 variants infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than wild-type.Abstract Figure

Background Epidermal growth factor receptor exon 20 insertion (EGFRex20ins) mutations represent approximately 4–12% of EGFR mutations and are generally refractory to the 1st and 2nd generation EGFR tyrosine kinase inhibitors (TKIs). Development of effective therapies for patients with EGFRex20ins mutant non-small-cell lung carcinoma (NSCLC) represents a great unmet need. Preclinical models have shown that osimertinib is active in NSCLC harboring EGFRex20ins, while the antitumor activity of osimertinib remains to be evaluated in patients with EGFRex20ins mutations. Methods Tumor genotyping was performed in 2316 Chinese NSCLC cases with targeted next generation sequencing (NGS) covering the whole exons of EGFR gene. The frequency and genetic characteristics of EGFRexon20ins mutations were analyzed. Furthermore, six patients with specific EGFRexon20ins mutations and receiving osimertinib 80 mg once daily were retrospectively included to assess the antitumor activity and safety of monotherapy osimertinib. Results EGFRex20ins mutations were identified in 4.8% (53/1095) of EGFR mutant NSCLC and 2.3% (53/2316) of all NSCLC cases. The most frequently identified EGFRexon20ins is A767_V769dup (17/53,32.1%). We found that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Four patients with osimertinib therapy achieved partial response and the rest stable disease. Median progression free survival (PFS) was 6.2 months (95% confidence interval 5.0–12.9 months; range 4.9–14.6 months). The most common adverse events (AEs) were diarrhea (2/6), pruritis (2/6), stomatitis (1/6) and nausea (1/6). No grade 3 or more AEs were documented. Conclusions This study revealed that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Furthermore, our study firstly demonstrated promising antitumor activity of osimertinib in certain EGFRex20ins mutant advanced NSCLC patients, indicating that osimertinib treatment for EGFRex20ins positive patients deserves further study. Electronic supplementary material The online version of this article (10.1186/s12885-019-5820-0) contains supplementary material, which is available to authorized users.

Background Many studies have investigated the role of microRNA-25 (miR-25) in the initiation and progression of sepsis in newborns. In this study, we aim to explore how rs41274221 polymorphism in miR-25 compromises the interaction between miR-25 and CD69, so as to understand the mechanisms involved in the control of sepsis in newborns. Methods Computational analysis, luciferase assay, real-time polymerase chain reaction (PCR), and western blot analysis were performed in this study. Results The luciferase assays results showed that CD69 was a target gene of miR-25, because the luciferase activity in cells transfected with wild type CD69 was much lower than that in the cells transfected with mutant CD69 or the scramble control. Real-time PCR and western blot analysis results showed that the expression of miR-25 in sepsis patients was significantly upregulated as compared with that in the normal control group, and the CD69 position ratio as well as the messenger RNA (mRNA) and protein level of CD69 in sepsis patients was much higher than those in the normal control group. As compared with the scramble control, miR-25 mimics, and CD69 small interfering RNA (siRNA) downregulated the mRNA and protein expression of CD69, whereas the expression of CD69 mRNA and protein in cells transfected with miR-25 inhibitors was significantly higher as compared with that in the scramble control. In addition, interferonγ production was significantly downregulated in cells transfected with miR-25 inhibitors but notably upregulated in cells transfected with miR-25 mimics or CD69 siRNA. Conclusion The single-nucleotide polymorphism (SNP; rs41274221) in miR-25 is associated with the risk of sepsis in newborns.

Hepatocellular carcinoma (HCC) usually occurs at the late stage of chronic liver injury. Oncostatin M (OSM) is a tumor-associated cytokine highly expressed in cirrhosis and HCC patients; however, its role in hepatocarcinogenesis has not been clearly elucidated. In this study, we investigated the effect of OSM on HCC occurrence in a rat model of N-diethylnitrosamine-induced HCC. OSM overexpression significantly increased the number of tumor nodules and shortened the overall survival of the rats. Notably, OSM promoted HPC activation in vivo but did not directly regulate the proliferation of the HPC cell line in vitro. Further, OSM induced tumor necrosis factor-α (TNF-α) secretion and CD68+ macrophage accumulation, which were positively correlated with HPC activation. Additionally, TNF-α or macrophage depletion inhibited the promoting effect of OSM on hepatocarcinogenesis and HPC activation. Furthermore, OSM expression in the peritumoral tissues of HCC was positively correlated with poor overall survival of patients. In conclusion, OSM plays an important role in hepatocarcinogenesis by regulating the liver inflammation environment. Hence, OSM could be used as a potential target for HCC prevention and therapy or as an indicator of HCC prognosis.

The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4–3.9 A. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaF2E2B2D6, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.

Achyranthes bidentata is a traditional Chinese medicine used to treat osteoporosis. AB90, a crude saccharide from A. bidentata, showed excellent osteoprotective effects in ovariectomized rats, and ABW90-1, an oligosaccharide purified from AB90, stimulated significant differentiation of osteoblasts. However, the osteogenic effects and underlying mechanisms of ABW90-1 have remained unknown. In the present study, we found that ABW90-1 significantly promoted ALP activity, mineralization, and the expression of osteogenic markers in MC3T3-E1 cells. ABW90-1 showed strong binding with the WNT signaling complex and BMP2 based on number of interactions, hydrogen bond length, and binding energy in silico. ABW90-1 significantly increased the expression of active-β-catenin, p-GSK-3β, LEF-1, BMP2, and p-SMAD1. Importantly, the osteogenic effects of ABW90-1 were partially suppressed by DKK-1 and Noggin, which are specific inhibitors of the WNT and BMP signaling pathways, respectively. Collectively, these findings suggest that ABW90-1 has osteogenic effects through crosstalk between WNT/β-catenin and BMP2/SMAD1 signaling pathways.

Background Several mechanisms including abnormal activation of PI3K-AKT-mTOR pathway have been proved to generate acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). In this study, we investigated the genomic characteristics of PI3K pathway activated in NSCLC patients after progression on EGFR-TKIs and whether both targeting EGFR and PI3K pathway could overcome resistance. Methods A total of 605 NSCLC cases with a history of EGFR TKI treatment were reviewed, in which 324 patients harboring EGFR mutations were confirmed progression on at least one EGFR TKI and finally enrolled. Tumor tissues or blood samples were collected at the onset of TKI progression for next generation sequencing (NGS). Six EGFR mutant patients with co-occurring mutations in PI3K pathway were retrospectively collected to assess the effect of EGFR TKI plus everolimus, a mTOR inhibitor. Results Forty-nine (14.9%) patients resistant to EGFR TKIs have at least one genetic variation in PI3K pathway. PIK3CA, PTEN and AKT1 variations were detected in 31 (9.5%), 18 (5.5%) and 3 (0.9%) of patients, respectively. No significant differences were observed in distribution of PI3K pathway alterations among patients with different EGFR mutations (EGFR exon19 deletion mutations/EGFR L858R/uncommon EGFR mutations) and among patients resistant to different EGFR TKIs. For patients treated with everolimus and EGFR-TKI, five (5/6, 83.3%) achieved stable disease (SD) and one (1/6, 16.7%) didn't receive disease control. The median progression-free survival (PFS) was 2.1 months (95% confidence interval, 1.35-4.3 months, range, 0.9-4.4 months). The most common adverse events were dental ulcer (6/6), rash (1/6). Conclusions Our study revealed that PI3K pathway was activated in at least 14.9% in EGFR-TKI resistant patients. EGFR-TKIs plus everolimus showed limited antitumor activity in EGFR mutant NSCLC patients with PI3K pathway aberrations.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution, the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that currently comprises seven different types based on structural homology in the TMDs.

Background: The development of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment paradigms for non-small cell lung cancer (NSCLC) patients. However, acquired resistance inevitably develops with a median progression-free survival (PFS) less than a year. The advancements in sequencing technologies have significantly promoted the identification of potential resistant mechanisms to targeted therapies.Case presentation: A 39-year-old Chinese male was diagnosed stage IVB NSCLC with EGFR exon 19 deletion (EGFR 19del) and received icotinib but got primary EGFR-TKI resistance caused by ROS1 fusion one month later. Then he received icotinib plus crizotinib and achieved partial response with a profession free survival of 4.8 months. Then he switched to osimertinib plus crizotinib due to the emergence of EGFR T790M mutation. TRIM24-BRAF fusion was detected after osimertinib failure, which might be potential resistant mechanism to osimertinib.Conclusions: This is the first case to confirm crizotinib plus icotinib in patients harboring both EGFR 19del and ROS1 fusion. This case displays the temporal heterogeneity of different resistant mechanisms emerging during disease course, highlighting the importance of dynamic genetic monitoring for better clinical managements.

Curli play critical roles in biofilm formation, host cell adhesion, and colonization of inert surfaces in many Enterobacteriaceae. In Escherichia coli, curli biogenesis requires 7 curli-specific gene (csg) products—CsgA through G—working in concert. Of them, CsgG and CsgF are 2 outer membrane (OM)-localized components that consists of the core apparatus for secretion and assembly of curli structural subunits, CsgB and CsgA. Here, we report the cryogenic electron microscopy (cryo-EM) structure of CsgG in complex with CsgF from E. coli. The structure reveals that CsgF forms a stable complex with CsgG via a 1:1 stoichiometry by lining the upper lumen of the nonameric CsgG channel via its N-terminal 27 residues, forming a funnel-like entity plugged in the CsgG channel and creating a unique secretion channel with 2 constriction regions, consistent with the recently reported structure of the CsgG-CsgF complex. Functional studies indicate that export of CsgF to the cell surface requires the CsgG channel, and CsgF not only functions as an adaptor that bridges CsgB with CsgG but also may play important roles in controlling the rates of translocation and/or polymerization for curli structural subunits. Importantly, we found that a series of CsgF-derived peptides are able to efficiently inhibit curli production to E. coli when administrated exogenously, highlighting a potential strategy to interfere biofilm formation in E. coli strains., Curli are functional amyloids that play critical roles in biofilm formation and adhesion in many Enterobacteriaceae. Two outer membrane proteins, CsgG and CsgF, form the core apparatus for secretion and assembly of the curli structural subunits. The cryo-EM structure of the CsgG-CsgF complex reveals a unique amyloid secretion channel with two tandem constriction regions.

Significance The ethylene signaling pathway has been extensively investigated in Arabidopsis, and EIN2 is the central component. Rice is a monocotyledonous model plant that exhibits different features in many aspects compared with the dicotyledonous Arabidopsis. Thus, rice provides an alternative system for identification of novel components of ethylene signaling. In this study, we identified a stabilizer of OsEIN2 through analysis of the rice ethylene-insensitive mutant mhz3. We found that MHZ3 stabilizes OsEIN2 likely by binding to its Nramp-like transmembrane domain and impeding protein ubiquitination, blocking proteasome-mediated protein degradation. This study reveals that MHZ3 is required for ethylene signaling and identifies how MHZ3 binds to OsEIN2 via the OsEIN2 N-terminal Nramp-like domain., The phytohormone ethylene regulates many aspects of plant growth and development. EIN2 is the central regulator of ethylene signaling, and its turnover is crucial for triggering ethylene responses. Here, we identified a stabilizer of OsEIN2 through analysis of the rice ethylene-response mutant mhz3. Loss-of-function mutations lead to ethylene insensitivity in etiolated rice seedlings. MHZ3 encodes a previously uncharacterized membrane protein localized to the endoplasmic reticulum. Ethylene induces MHZ3 gene and protein expression. Genetically, MHZ3 acts at the OsEIN2 level in the signaling pathway. MHZ3 physically interacts with OsEIN2, and both the N- and C-termini of MHZ3 specifically associate with the OsEIN2 Nramp-like domain. Loss of mhz3 function reduces OsEIN2 abundance and attenuates ethylene-induced OsEIN2 accumulation, whereas MHZ3 overexpression elevates the abundance of both wild-type and mutated OsEIN2 proteins, suggesting that MHZ3 is required for proper accumulation of OsEIN2 protein. The association of MHZ3 with the Nramp-like domain is crucial for OsEIN2 accumulation, demonstrating the significance of the OsEIN2 transmembrane domains in ethylene signaling. Moreover, MHZ3 negatively modulates OsEIN2 ubiquitination, protecting OsEIN2 from proteasome-mediated degradation. Together, these results suggest that ethylene-induced MHZ3 stabilizes OsEIN2 likely by binding to its Nramp-like domain and impeding protein ubiquitination to facilitate ethylene signal transduction. Our findings provide insight into the mechanisms of ethylene signaling.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins represents a unique membrane protein family that facilitates the insertion, folding, and assembly of a cohort of α-helical membrane proteins in all kingdoms of life, yet its underlying mechanisms remain elusive. We report the crystal structures of the full-length Thermotoga maritima YidC (TmYidC) and the TmYidC periplasmic domain (TmPD) at a resolution of 3.8 and 2.5 A, respectively. The crystal structure of TmPD reveals a β-supersandwich fold but with apparently shortened β strands and different connectivity, as compared to the Escherichia coli YidC (EcYidC) periplasmic domain (EcPD). TmYidC in a detergent-solubilized state also adopts a monomeric form and its conserved core domain, which consists of 2 loosely associated α-helical bundles, assemble a fold similar to that of the other YidC homologues, yet distinct from that of the archaeal YidC-like DUF106 protein. Functional analysis using in vivo photo-crosslinking experiments demonstrates that Pf3 coat protein, a Sec-independent YidC substrate, exits to the lipid bilayer laterally via one of the 2 α-helical bundle interfaces: TM3-TM5. Engineered intramolecular disulfide bonds in TmYidC, in combination with complementation assays, suggest that significant rearrangement of the 2 α-helical bundles at the top of the hydrophilic groove is critical for TmYidC function. These experiments provide a more detailed mechanical insight into YidC-mediated membrane protein biogenesis.-Xin, Y., Zhao, Y., Zheng, J., Zhou, H., Zhang, X. C., Tian, C., Huang, Y. Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF-2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF-2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β-glycerophosphate). FGF-2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type I, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF-2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT-qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF-2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF-2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Calcium ions (Ca2+) are a major constituent of most pulp-capping materials and have an important role in the mineralization of human dental pulp cells (hDPCs). A previous study by our group has shown that increased levels of Ca2+ can promote hDPC-mediated mineralization in long-term cultures (21 days). However, the initiation of mineralization occurs in the early stage of osteogenic inductive culture, and the effects of Ca2+ on the mineralization of hDPCs in short-term cultures (five days) have not been studied in detail. Furthermore, the underlying mechanism by which Ca2+ stimulates the mineralization of hDPCs has remained controversial. A strong correlation between mineralization and cell apoptosis and/or death has been identified. Thus, the present study hypothesized that Ca2+ may promote the onset of hDPC-mediated mineralization through inducing their apoptosis and/or death. To verify this hypothesis, Ca2+ was added to the growth culture medium and osteogenic culture medium at various concentrations. Alizarin Red S staining and reverse transcription-polymerase chain reaction analysis were used to evaluate the onset of mineralization. Furthermore, the cell counting kit-8 and fluorescein isothiocyanate-Annexin V/propidium iodide double-staining method were adopted to detect the proliferation and apoptosis of hDPCs in the growth culture medium. An animal experiment and scanning electron microscopic observation of ceramic graft implants were applied to measure the mineralization in vivo. The results showed that 5.4 and 9.0 mM Ca2+ accelerated the onset of mineralized matrix nodule formation, promoted osteopontin mRNA expression and induced marked cell apoptosis and necrosis, but had no obvious effect on cell proliferation. These findings indicated a positive association between cell apoptosis and/or death and the timing of formation as well as the quantity of extracellular mineralization induced by Ca2+ in short-term cultured hDPCs.

Introduction Adseverin is an actin-severing and actin-capping protein that is primarily expressed in secretory cells, where it regulates the filamentous actin cytoskeleton during cell differentiation and exocytosis. However, little is known regarding its regulatory role in dental pulp cells (DPCs). This study examined the expression and function of adseverin in the proliferation, migration, and odontoblastic differentiation of DPCs. Methods DPCs were assayed for morphologic changes, proliferation, migration, alkaline phosphatase activity, and dentin sialoprotein and dentin matrix protein-1 protein levels in vitro after knockdown of adseverin by using small interfering RNA. Tooth germs isolated from Sprague-Dawley rats were processed for immunohistochemistry analysis of adseverin. Results Adseverin expression was increased in a time-dependent fashion in the early stage of odontoblastic differentiation. When adseverin expression was suppressed in DPCs, their cellular morphology was altered, and their proliferation, migration, and odontoblastic differentiation were substantially decreased in vitro . Secretory odontoblasts in the tooth germ at day 5 post partum expressed a stronger adseverin signal compared with those at days 1 and 3 post partum. Conclusions Adseverin may play a crucial role in the proliferation, migration, and odontoblastic differentiation of DPCs via filamentous actin cytoskeleton regulation. However, further investigations are required to clarify the underlying mechanisms.

Lipopolysaccharide (LPS) is one of the causative agents of pulpitis and previous studies have demonstrated that the LPS stimulation of human aortic valve interstitial cells induces inflammatory mediators and the gene expression of osteogenic factors. Therefore, in the present study, it was hypothesized that LPS affects the odontoblastic differentiation of human dental pulp cells (hDPCs). In order to investigate this, an in vitro study using hDPCs was performed. Increased alkaline phosphatase (ALP) activity was observed in the hDPCs treated with LPS, which was more marked when the cells were costimulated with odontogenic induction medium (OM). LPS also appeared to increase the gene expression levels of dentin sialophosphoprotein and dentin matrix protein‑1 and the protein expression level of dental sialoprotein in the hDPCs, particularly in combination with OM. In addition, the size and the number of nodules formed in the hDPCs exposed to OM and LPS were increased compared to those stimulated by OM alone. To determine the role of nuclear factor κB (NF‑κB) during the LPS‑induced odontoblastic differentiation of hDPCs, immunofluorescence was performed. The nuclear translocation of NF‑κB, induced by LPS was confirmed, suggesting its involvement in the LPS‑induced increase in odontoblastic differentiation of hDPCs. In conclusion, there may be an association between LPS stimulation, with or without OM, and odontoblastic differentiation.

High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

Lipopolysaccharide, an essential component of the Gram-negative bacteria outer membrane, is inserted by LptD–LptE, a protein complex with a unique ‘barrel and plug’ architecture; the structure of the LptD–LptE complex of Shigella flexneri determined here shows LptD forming a 26-stranded β-barrel with LptE located inside the barrel of LptD, the first two β-strands are distorted by two proline residues, creating a potential portal in the barrel wall that might allow lateral diffusion of lipopolysaccharide into the outer membrane. Haohao Dong et al. and Shuai Qiao et al. present X-ray crystal structures of the complex between the lipopolysaccharide transport proteins LptD and LptE from the bacteria Salmonella typhimurium and Shigella flexneri, respectively. The two papers report a unique two-protein plug-and-barrel architecture for the LptD–LptE complex that reveals the mechanism by which the cell-wall lipopolysaccharide is delivered and inserted into the external leaflet of the outer membrane of Gram-negative bacteria. As well as providing new detail on the nature of outer membrane biogenesis, this work will provide data relevant to the design of new antibiotic strategies targeting the bacterial outer membrane, much needed in the fight against multi-drug resistant pathogens. One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS)1. The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface2. Of the seven Lpt proteins, the LptD–LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane3,4. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD–LptE from Shigella flexneri at 2.4 A resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded β-barrel formed by LptD. Importantly, the secondary structures of the first two β-strands are distorted by two proline residues, weakening their interactions with neighbouring β-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD–LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.

Background EGFR-TKIs have shown remarkable effect in non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. Nevertheless, several mechanisms including activation of PI3K/AKT/mTOR pathway have been proved to generate acquired assistance to EGFR-TKIs. In this study, we investigated the genomic characteristics of PI3K pathway in NSCLC patients with acquired resistance to EGFR-TKIs and whether both targeting EGFR and mTOR could reverse resistance. Methods A total of 605 NSCLC cases who received prior TKI treatment were reviewed, in which 328 patients harboring EGFR-mutants were confirmed to have progressed on one or more TKI and finally enrolled. FFPE tumor or blood samples were collected at the onset of TKI resistance for NGS based panel assay (PIK3CA/PTEN/AKT1 genes and NSCLC driver genes are included). Six patients with co-mutations in EGFR and PI3K pathway who had been heavily treated and progressed on EGFR-TKI received EGFR-TKI plus mTOR inhibitor everolimus combination therapy. Results In a total of 328 patients, 294 (89.6%) of the patients were adenocarcinomas, 4(12.2%) were small cell lung cancer, while the rest were squamous carcinoma or NSCLC not otherwise specified (30, 9.2%). Forty nine (14.9%) patients with TKI resistant have genomic alterations in PI3K pathway. PIK3CA, PTEN and AKT1variations were detected in 31(9.5%), 18(5.5%) and 3(0.9%) of patients respectively. Three patients had both PIK3CA and PTEN mutations and one patient had co-mutation of PIK3CA/PTEN/AKT1. PIK3CA hotspot mutations of E545, H1047 and E542 accounted for 60.5% of PIK3CA mutations. Six patients were treated with everolimus (5mg) plus EGFR-TKI (2 patients with gefitinib, 250mg; 1 patient with afatinib, 30mg; 3 patients with osimertinib 80mg, daily) combination therapy. One (1/6, 16.7%) patient achieved partial response and the rest (5/6, 83.3%) achieved stable disease. The most common adverse events were dental ulcer (6/6), rash (2/6), pruritis (1/6) and diarrhea (1/6). One patient withdrew combination treatment due to grade 3 dental ulcer. Conclusions Our study revealed that PI3K pathway was activated in approximately 15% of EGFR-TKI resistant patients. Combination therapy of EGFR-TKIs and everolimus revealed limited antitumor activity in EGFR-TKI resistant NSCLC patients with dysregulation of PI3K pathway. Citation Format: Wenfeng Fang, Yihua Huang, Jiadi Gan, Yunpeng Yang, Yifen Wu, Jingbin Huang, Zhiyong Xu, Wenjing Wang, Li Zhang. The impact of PIK3CA/PTEN/AKT1 genes in advanced NSCLC patients with acquired EGFR-TKI resistance and clinical response to EGFR-TKI plus everolimus combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 327.

In Gram-negative bacteria, the assembly of β-barrel outer-membrane proteins (OMPs) requires the β-barrel-assembly machinery (BAM) complex. We determined the crystal structure of the 200-kDa BAM complex from Escherichia coli at 3.55-A resolution. The structure revealed that the BAM complex assembles into a hat-like shape, in which the BamA β-barrel domain forms the hat's crown embedded in the outer membrane, and its five polypeptide transport-associated (POTRA) domains interact with the four lipoproteins BamB, BamC, BamD and BamE, thus forming the hat's brim in the periplasm. The assembly of the BAM complex creates a ring-like apparatus beneath the BamA β-barrel in the periplasm and a potential substrate-exit pore located at the outer membrane-periplasm interface. The complex structure suggests that the chaperone-bound OMP substrates may feed into the chamber of the ring-like apparatus and insert into the outer membrane via the potential substrate-exit pore in an energy-independent manner.

In addition to caspase inhibition, X-linked inhibitor of apoptosis (XIAP) induces NF-kappaB and MAP kinase activation during TGF-b and BMP receptor signaling and upon overexpression. Here we show that the BIR1 domain of XIAP, which has no previously ascribed function, directly interacts with TAB1 to induce NF-kappaB activation. TAB1 is an upstream adaptor for the activation of the kinase TAK1, which in turn couples to the NF-kappaB pathway. We report the crystal structures of BIR1, TAB1, and the BIR1/TAB1 complex. The BIR1/TAB1 structure reveals a striking butterfly-shaped dimer and the detailed interaction between BIR1 and TAB1. Structure-based mutagenesis and knockdown of TAB1 show unambiguously that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-kappaB activation. We show that although not interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibits the XIAP/TAB1 interaction. Disruption of BIR1 dimerization abolishes XIAP-mediated NF-kappaB activation, implicating a proximity-induced mechanism for TAK1 activation.

The interaction between Arabidopsis thaliana and the oomycete pathogen Phytophthora parasitica emerges as a model for exploring the molecular basis and evolution of recognition and host defense. Phenotypic variation and genetic analysis is essential to dissect the underlying mechanisms in plant-oomycete interaction. In this study, the reaction phenotypes of 28 A. thaliana accessions to P. parasitica strain Pp016 were examined using detached leaf infection assay. The results showed the presence of four distinct groups based on host response and disease development. Of all the accessions examined, Zurich (Zu-1) is highly resistant to P. parasitica. Microscopic characterization showed that rapid and severe hypersensitive response (HR) at the primary infection epidermal cells is associated with disease resistance. Furthermore, Zu-1 is resistant to a set of 20 diverse P. parasitica strains, which were collected from different host plants and exhibited differential specificities on a set of tobacco cultivars. However, Zu-1 is susceptible to P. parasitica when the root is inoculated, suggesting differential expression of associated resistance genes in the root and foliar tissues. Genetic analysis by crossing Zu-1 and the susceptible accession Landsberg (Ler) showed that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1Zu-1 (for Resistance to Phytophthora parasitica 1), as shown by analysis of F2 segregating populations. By employing specific-locus amplified fragment sequencing (SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1-11.2 Mb in chromosome IV. The future cloning of RPPA1Zu-1 locus will facilitate improved understanding of plant broad-spectrum disease resistance to oomycete pathogens.

In gram-negative bacteria, assembly of outer membrane proteins requires the multicomponent β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. To understand how BamA facilitates outer membrane protein biogenesis, it is important to obtain sufficient amounts of purified recombinant BamA protein for in vitro functional analysis and structure determination. In this chapter, we describe the protocol that we used in our laboratory for the cloning, expression, and purification of E. coli BamA and its N-terminal deletion variants for in vitro functional studies and for structure determination of the β-barrel domain alone (residues 426-810).

Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome- Drosophila, Arabidopsis, Synechocystis , Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Natl Acad Sci USA 103:17696–17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 Å resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-binding cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.

Various strains of bacteria are able to produce a unique class of functional amyloids termed curli, which are critical for biofilm formation, host cell adhesion, and colonization of inert surfaces. Curli are secreted via the type VIII bacterial secretion system, and they share biochemical and structural characteristics with amyloid fibers that have been implicated in deleterious disease in humans. Here, we report the crystal structure of Escherichia coli CsgG, which is an essential lipoprotein component of the type VIII secretion system and which forms a secretion channel in the bacterial outer membrane for transporting curli subunits. CsgG forms a crown-shaped, symmetric nonameric channel that spans the outer membrane via a 36-strand β-barrel, with each subunit contributing four β-strands. This nonameric complex contains a central channel with a pore located at the middle. The eyelet of the pore is ∼12 A in diameter and is lined with three stacked nine-residue rings consisting of Tyr-66, Asn-70, or Phe-71. Our structure-based functional studies suggest that Tyr-66 and Phe-71 residues function as gatekeepers for the selective secretion of curli subunits. Our study describes in detail, to our knowledge, the first core structure of the type VIII bacterial secretion machinery. Importantly, our structural analysis suggests that the curli subunits are secreted via CsgG across the bacterial outer membrane in an unfolded form.

In Gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a five-protein β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. Here, the refolding, crystallization and preliminary X-ray crystallo­graphic characterization of the β-barrel domain of BamA from Escherichia coli (EcBamA) are reported. Native and selenomethionine-substituted EcBamA proteins were crystallized at 16°C and X-ray diffraction data were collected to 2.6 and 3.7 A resolution, respectively. The native crystals belonged to space group P21212, with unit-cell parameters a = 118.492, b = 159.883, c = 56.000 A and two molecules in one asymmetric unit; selenomethionine-substituted protein crystals belonged to space group P4322, with unit-cell parameters a = b = 163.162, c = 46.388 A and one molecule in one asymmetric unit. Initial phases for EcBamA β-barrel domain were obtained from a SeMet SAD data set. These preliminary X-ray crystallographic studies paved the way for further structural determination of the β-barrel domain of EcBamA.

The inhibitor of apoptosis proteins (IAPs) represent the only endogenous caspase inhibitors and are characterized by the presence of baculoviral IAP repeats (BIRs). Here, we report the crystal structure of the complex between human caspase-7 and XIAP (BIR2 and the proceeding linker). The structure surprisingly reveals that the linker is the only contacting element for the caspase, while the BIR2 domain is invisible in the crystal. The linker interacts with and blocks the substrate groove of the caspase in a backward fashion, distinct from substrate recognition. Structural analyses suggest that the linker is the energetic and specificity determinant of the interaction. Further biochemical characterizations clearly establish that the linker harbors the major energetic determinant, while the BIR2 domain serves as a regulatory element for caspase binding and Smac neutralization.

Ushers constitute a family of bacterial outer membrane proteins responsible for the assembly and secretion of surface organelles such as the pilus. The structure at 3.15-Å resolution of the usher pyelonephritis-associated pili C (PapC) translocation domain reveals a 24-stranded kidney-shaped β-barrel, occluded by an internal plug domain. The dimension of the pore allows tandem passage of individual folded pilus subunits in an upright pilus growth orientation, but is insufficient for accommodating donor strand exchange. The molecular packing revealed by the crystal structure shows that 2 PapC molecules in head-to-head orientation interact via exposed β-strand edges, which could be the preferred dimer interaction in solution. In vitro reconstitution of fiber assemblies suggest that PapC monomers may be sufficient for fiber assembly and secretion; both the plug domain and the C-terminal domain of PapC are required for filament assembly, whereas the N-terminal domain is mainly responsible for recruiting the chaperone–subunit complexes to the usher. The plug domain has a dual function: gating the β-pore and participating in pilus assembly.

Genome-wide association (GWA) studies revealed a number of single nucleotide polymorphisms (SNPs) significantly associated with type 1 diabetes (T1D). In an attempt to confirm some of these candidate associations, we genotyped 2046 Caucasian patients and 2417 normal controls from the United States for SNPs in five genomic regions. While no evidence was obtained for four genomic regions (rs2929366/NM_144715 on chromosome 3, rs9127/Q7Z4C4 on chromosome 5, rs1445898/CAPSL on chromosome 5 and rs2302188/NM_033543 on chromosome 19), we provide strong evidence for association between T1D and multiple SNPs in the IFIH1 linkage disequilibrium (LD) block on chromosome 2q. Among the 10 SNPs genotyped for the 2q region, four SNPs located within the IFIH1 gene or at the 5' region of IFIH1 showed significant association with T1D in the Georgia population [odds ratio (OR) = 1.7-1.9] with the best P-value found at SNP rs1990760 (P = 8 x 10(-8) and OR = 1.9). Several SNPs outside of the IFIH1 gene also showed significant but weaker associations. Furthermore, IFIH1 gene expression levels in peripheral blood mononuclear cells are significantly correlated with IFIH1 genotypes, and higher IFIH1 levels are found in individuals with the susceptible genotypes (P = 0.005). Thus, both genetic association and gene expression data suggest that IFIH1 is the most plausible candidate gene implicated in T1D in this LD block.

X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms1,2. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition3,4,5,6,7. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

In this issue of Cancer Cell, Schimmer et al. report the identification of small molecule antagonists of XIAP that overcome its inhibition of caspase-3. It was remarkable that the compounds directly induced cell death in tumor cells while having little toxicity on normal cells. This suggests that caspases are already activated in tumor cells, which is different from the caspase activation status in normal mammalian cells. In comparison with Smac peptides targeting XIAP-mediated caspase-9 inhibition, which do not directly induce cell death, it appears that liberating downstream caspases rather than upstream caspases may be a preferred strategy for cancer drug discovery.