GPCR targeting of E3 ubiquitin ligase MDM2 by inactive ß-arrestin.

E3 ubiquitin ligase Mdm2 facilitates ß-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, ß-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the ß-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the ß-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of ß-arrestin1 in complex with Mdm2ABR peptide. The acidic residues of Mdm2ABR bind to the positively charged concave side of the ß-arrestin1 N-domain. The C-tail of ß-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of ß-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate ß-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on ß-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2ABR binding to ß-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP6-induced oligomer of ß-arrestin1. These results show how the E3 ligase, Mdm2, interacts with ß-arrestins to promote the internalization of GPCRs.

FOXL2 and FOXA1 cooperatively assemble on the TP53 promoter in alternative dimer configurations.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein-protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

Deciphering the Role of ERBB3 Isoforms in Renal Cell Carcinoma: A Comprehensive Genomic and Transcriptomic Analysis.

ERBB3, a key member of the receptor tyrosine kinase family, is implicated in the progression and development of various human cancers, affecting cellular proliferation and survival. This study investigated the expression of ERBB3 isoforms in renal clear cell carcinoma (RCC), utilizing data from 538 patients from The Cancer Genome Atlas (TCGA) Firehose Legacy dataset. Employing the SUPPA2 tool, the activity of 10 ERBB3 isoforms was examined, revealing distinct expression patterns in RCC. Isoforms uc001sjg.3 and uc001sjh.3 were found to have reduced activity in tumor tissues, while uc010sqb.2 and uc001sjl.3 demonstrated increased activity. These variations in isoform expression correlate with patient survival and tumor aggressiveness, indicating their complex role in RCC. The study, further, utilizes CIBERSORTx to analyze the association between ERBB3 isoforms and immune cell profiles in the tumor microenvironment. Concurrently, Gene Set Enrichment Analysis (GSEA) was applied, establishing a strong link between elevated levels of ERBB3 isoforms and critical oncogenic pathways, including DNA repair and androgen response. RT-PCR analysis targeting the exon 21-23 and exon 23 regions of ERBB3 confirmed its heightened expression in tumor tissues, underscoring the significance of alternative splicing and exon utilization in cancer development. These findings elucidate the diverse impacts of ERBB3 isoforms on RCC, suggesting their potential as diagnostic markers and therapeutic targets. This study emphasizes the need for further exploration into the specific roles of these isoforms, which could inform more personalized and effective treatment modalities for renal clear cell carcinoma.

Clinical Efficacy of Neoadjuvant Intravesical Mitomycin-C Therapy Immediately Before Transurethral Resection of Bladder Tumor in Patients With Nonmuscle-invasive Bladder Cancer: Preliminary Results of a Prospective, Randomized Phase II Study.

PURPOSE: Intravesical mitomycin-C is recommended immediately after transurethral resection of bladder tumor for nonmuscle-invasive bladder cancer. However, a lack of compliance occurs due to the associated complications. Here, we aimed to assess the efficacy and safety of intravesical mitomycin-C before transurethral resection of bladder tumor in patients with nonmuscle-invasive bladder cancer. MATERIALS AND METHODS: This was a single-center, open-label, parallel-arm, randomized phase II clinical trial in patients with suspected nonmuscle-invasive bladder cancer before transurethral resection of bladder tumor. Participants were randomly assigned (1:1) to receive 2 doses of intravesical mitomycin-C (40 mg/20 mL) 1 day and 4 hours before transurethral resection of bladder tumor (n = 49) or no treatment (n = 50) with block randomization (size 2 and 4), stratified by bacillus Calmette-Guérin/intravesical mitomycin-C. The primary endpoint was recurrence-free survival and secondary endpoints were progression-free survival and adverse events in the per-protocol analysis. RESULTS: Seventy-one patients (33, intervention; 38, control) were well matched for baseline characteristics. Sixty-one had been followed without recurrence for at least 10.4 months; 3 and 8 patients showed recurrence in the intervention and control groups, respectively. The 1-year recurrence-free survival rate was 97% and 89% for the intervention and control groups, respectively. Neoadjuvant intravesical mitomycin-C resulted in a reduction (63%) in the relative recurrence risk (hazard ratio, 0.37; 80% 1-sided confidence interval, -∞-0.65, P = .11). Disease progression occurred in 3 patients in the control group (P = .051) but not in the intervention group. Neoadjuvant intravesical mitomycin-C was well tolerated, and adverse events were local and of grade 1/2. CONCLUSIONS: Two doses of neoadjuvant intravesical mitomycin-C are safe and effective in reducing nonmuscle-invasive bladder cancer recurrence and progression after transurethral resection of bladder tumor.

Inhibition mechanism of testis-expressed gene 14 (TEX14) in cytokinetic abscission: Well-tempered metadynamics simulation studies.

Cytokinesis requires a apoptosis-linked gene 2 interacting protein X (ALIX) and a 55 kDa midbody centrosomal protein (CEP55) to activate the cell abscission in somatic cells. However, in germ cells, CEP55 forms intercellular bridges with testis-expressed gene 14 (TEX14), which blocks the cell abscission. These intercellular bridges play important roles in the synchronization of the germ cells and facilitate the coordinated passage of organelles and molecules between germ cells. If TEX14 is intentionally removed, intercellular bridges are disrupted, leading to sterility. Hence, a deeper understanding regarding the roles of TEX14 can provide significant insights into the inactivation of abscission and the inhibition of proliferation in cancer cells. Previous experimental studies have shown that the high affinity and low dissociation rate of TEX14 for CEP55 prevent ALIX from binding CEP55 and inactivate the germ cell abscission. However, detailed information about how TEX14 interacts with CEP55 to prevent the cell abscission is still lacking. To gain more specific insights into the interactions between CEP55 and TEX14 and the difference in reactivity between TEX14 and ALIX, we performed well-tempered metadynamics simulations of these protein complexes using atomistic models of CEP55, TEX14, and ALIX. We identified the major binding residues of TEX14 and ALIX with CEP55 by using 2D Gibbs free energy evaluations, the results of which are consistent with previous experimental studies. Our results may help design synthetic TEX14 mimicking peptides, which can bind CEP55 and facilitate the inactivation of abscission in abnormal cells, including cancer cells.

Surgical outcomes of ureteral reconstruction during cytoreductive surgery for ovarian cancer: a retrospective cohort study.

BACKGROUND: Ureteral reconstruction is required after surgical resection of the tumor invading the urinary tract in ovarian cancer with low incidence. There are no currently reported surgical outcomes of ureteral reconstruction during cytoreductive surgery. The aim of the study is to investigate the clinical features and surgical outcomes of ureteral reconstruction during primary, interval and secondary cytoreductive surgery for ovarian cancer.  METHODS: A total of 3226 patients who underwent primary, interval or secondary cytoreductive surgery for ovarian cancer between January 2000 and May 2021 were reviewed. Fifty-six patients who underwent ureteral reconstruction during cytoreductive surgery were included in the analysis.  RESULTS: Ureteral reconstruction was required in 1.7% (56/3226) of ovarian cancer patients. Of the 56 patients who underwent ureteral reconstruction during cytoreductive surgery, 35 (62.5%) had primary ovarian cancer, and 21 (37.5%) had recurrent ovarian cancer. The median tumor size invading the lower urinary tract was 2.0 cm (range, 0.4-9.5 cm). Ureteroneocystostomy with direct implantation (51.8%) and psoas hitch (8.9%), transureteroureterostomy (7.1%), and ureteroureterostomy (32.1%) were required as part of cytoreductive surgery. Complete cytoreduction with ureteral reconstruction was achieved in 83.9% (47/56) and the rest of the patient population (16.1%) achieved a gross residual tumor size of less than 1 cm. All complications, including hydronephrosis (33.9%), were managed, none resulting in long-term sequelae. In primary ovarian cancer, the 5-year disease-free survival and overall survival were 50.0% and 89.5%, respectively. In patients with recurrent ovarian cancer, the 5-year disease-free survival and overall survival were 23.6% and 64.0%, respectively. CONCLUSIONS: Ureteral reconstruction as a part of cytoreductive surgery for ovarian cancer could be performed with acceptable morbidities. Complete cytoreduction by a multidisciplinary surgical team, including urologic oncologists, should be pursued for the surgical management of ovarian cancer. TRIAL REGISTRATION: Retrospectively registered.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Thermodynamic Models for Assembly of Intrinsically Disordered Protein Hubs with Multiple Interaction Partners.

Prevalent in diverse protein interactomes, intrinsically disordered proteins or regions (IDPs or IDRs) often drive assembly of higher-order macromolecular complexes, using multiple target-binding motifs. Such IDP hubs are suggested to process various cellular signals and coordinate relevant biological processes. However, the mechanism of assembly and functional regulation of IDP hubs remains elusive due to the challenges in dissecting their intricate protein-protein interaction networks. Here we present basic thermodynamic models for the assembly of simple IDP hubs with multiple target proteins, constructing partition functions from fundamental binding parameters. We combined these basic functions to develop advanced thermodynamic models to analyze the assembly of the Nup153 hubs interacting with multiple karyopherin ß1 (Kap) molecules, critical components of nucleocytoplasmic transport. The thermodynamic analysis revealed a complex organization of the Kap binding sites within the C-terminal IDR of Nup153 including a high-affinity 1:1 interaction site and a series of low-affinity sites for binding of multiple Kaps with negative cooperativity. The negative cooperativity arises from the overlapping nature of the low-affinity sites where Kap occupies multiple dipeptide motifs. The quantitative dissection culminated in construction of the Nup153 hub ensemble, elucidating how distribution among various Kap-bound states is modulated by Kap concentration and competing nuclear proteins. In particular, the Kap occupancy of the IDR can be fine-tuned by varying the location of competition within the overlapping sites, suggesting coupling of specific nuclear processes to distinct transport activities. In general, our results demonstrate the feasibility and a potential mechanism for manifold regulation of IDP functions by diverse cellular signals.

Molecular dynamics study with mutation shows that N-terminal domain structural re-orientation in Niemann-Pick type C1 is required for proper alignment of cholesterol transport.

The lysosomal membrane protein Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) are main players of cholesterol control in the lysosome and it is known that the mutation on these proteins leads to the cholesterol trafficking-related neurodegenerative disease, which is called the NPC disease. The mutation R518W or R518Q on the NPC1 is one of the type of disease-related mutation that causes cholesterol transports to be cut in half, which results in the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment of the cell. Even though there has been significant progress with understanding the cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of the full-length NPC1 in 2016, many details such as the interaction of the full-length NPC1 with the NPC2, the molecular motions responsible for the cholesterol transport during and after this interaction, and the structure and the function relations of many mutations are still not well understood. In this study, we report the extensive molecular dynamics simulations in order to gain insight into the structure and the dynamics of NPC1 lumenal domain for the cholesterol transport and the disease behind the mutation (R518W). It was found that the mutation induces a structural shift of the N-terminal domain, toward the loop region in the middle lumenal domain, which is believed to play a central role in the interaction with NPC2 protein, so the interaction with the NPC2 protein might be less favorable compared to the wild NPC1. Also, the simulation indicates the possible re-orientation of the N-terminal domain with both the wild and the R518W-mutated NPC1 after receiving the cholesterol from the NPC2 that align to form an internal tunnel, which is a possible pose for further action in cholesterol trafficking. We believe the current study can provide a better understanding of the cholesterol transport by NPC1 especially the role of NTD of NPC1 in combination with NPC2 interactions.

Crystal structure of proteolyzed VapBC and DNA-bound VapBC from Salmonella enterica Typhimurium LT2 and VapC as a putative Ca2+ -dependent ribonuclease.

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.

The effect of subsequent immunosuppressant use in organ-transplanted patients on prostate cancer incidence: a retrospective analysis using the Korean National Health Insurance Database.

BACKGROUNDS: Prostate cancer (PC) is the most common solid organ cancer. However, there is still no definite consensus before and after organ transplantation (TPL). We aimed to analyze whether PC incidence increased in TPL patients with subsequent use of immunosuppressants using the Korean National Health Insurance Database. METHODS: TPL patients between 2003 and 2015(N = 12,970) were age- and year-matched to non-TPL patients (N = 38,910) in a 1:3 ratio. Multivariate Cox regression analysis adjusted for significant prognostic clinicopathological parameters, including the duration of immunosuppressant agent use (0-300 or > 300 days), and Kaplan-Meier analysis with log-rank test were used to evaluate the association of TPL with PC incidence between the groups. RESULTS: Median overall survival was 4.86 years; overall mortality rate was 3.4% (n = 1761). Regardless of differences in baseline characteristics between the groups, multivariate analysis for PC incidence showed that age, immunosuppressant use, and TPL organ subtypes were significant factors for the overall population, whereas only age was significant in the TPL group (p < 0.05). After adjusting for age, underlying disease, and prescribed medication (aspirin, statin), multiple subgroup analysis models for PC incidence were evaluated. PC incidence was increased in the TPL group (hazard ratio [HR] 1.965, p < 0.001); however, PC incidence in the TPL group became insignificant after adjusting for immunosuppressant use (p = 0.194). Kaplan-Meier curves also showed that PC incidence was significantly different according to age and TPL with the use of immunosuppressants between the TPL and non-TPL groups. CONCLUSIONS: PC incidence was higher in the TPL group using immunosuppressants than in the non-TPL group. TRIAL REGISTRATION:  The study was retrospectively registered.

Credé maneuver to adjust tape tension during trans-obturator tape mid-urethral sling in mixed urinary incontinence.

INTRODUCTION AND HYPOTHESIS: To evaluate the efficacy of intraoperative extrinsic manual bladder compression (Credé maneuver) for trans-obturator tape adjustment during mid-urethral sling surgery in women with stress urinary incontinence and those with mixed urinary incontinence. METHODS: The study included 148 randomly selected women who underwent mid-urethral sling surgery with trans-obturator tape for stress urinary incontinence between January 2016 and May 2017. Subgroup analysis of 66 women with mixed urinary incontinence included 43 patients from the Credé maneuver group and 23 from the non-Credé maneuver group. In the Credé maneuver group, the pattern of urine leakage was determined during the Credé maneuver, and tape tension was adjusted according to the pattern. RESULTS: The cure rate was 86.6% and improved rate was 11.9% in the Credé maneuver patients. The cure rate was 50.6% and improved rate was 38.3% in the non-Credé maneuver patients. The success rate was significantly higher in the Credé than in the non-Credé maneuver group (p = 0.023). In subgroup analysis of patients with mixed urinary incontinence, the cure rate was 81.4% and improved rate was 16.3% in the Credé maneuver group. The cure rate was 43.5% and improved rate was 47.8% in the non-Credé maneuver group. The cure rate was significantly higher in the Credé maneuver group (p = 0.007). CONCLUSIONS: Intraoperative trans-obturator tape adjustment using the Credé maneuver to identify the leaking pattern significantly improved the success rate in women with mixed urinary incontinence, and Credé maneuver-directed adjustment significantly improved the cure rate.

Phosphatidylserine-induced dissociation of the heterodimeric PstB2p/Pbi1p complex in yeast phosphatidylserine trafficking system.

Most phospholipids-essential building blocks of cellular membranes-are synthesized in the endoplasmic reticulum (ER) and distributed to the intracellular membranes. Yeast phosphatidylserine (PtdSer) is produced in the ER and is transported to the mitochondria, Golgi, or vacuoles; it is subsequently converted into phosphatidylethanolamine by phosphatidylserine decarboxylase. In yeast, PstB2p (Sec14p homolog) and Pbi1p are known to be involved in non-vesicular lipid transport from the ER to the Golgi, however, the molecular mechanisms remain unclear. In this study, we attempted to analyze the stoichiometric model of the PstB2p-Pbi1p complex in PtdSer transport from the ER to Golgi by using size exclusion chromatography with multi-angle static light scattering, mass spectrometry, reductive methylation, and homology modeling. The homology model of PstB2p was validated in part via reductive methylation, suggesting that it has structure similar to that of Sec14p. We observed that PstB2p forms a homodimer but exists as a 1:1 heterodimer in the presence of Pbi1p. When PtdSer was added to the PstB2p-Pbi1p complex, PtdSer bound to PstB2p, triggering the dissociation of the PstB2p-Pbi1p complex. PstB2p in complex with PtdSer exists as a monomer in contrast to its homodimeric form in the absence of PtdSer. These findings suggest that a stoichiometric model of the PstB2p-Pbi1p complex in yeast can be used to study the PtdSer transport system.

Structural characterization of a putative diguanylate cyclase conserved in hyperthermophiles.

C-di-GMP, bis-(3'-5')-cyclic dimeric guanosine monophosphate, is a key signaling molecule that regulates many important physiological processes in bacteria. C-di-GMP is synthesized by diguanylate cyclase (DGC) containing the homodimeric GGDEF domain. There are many uncharacterized hypothetical proteins annotated as a putative DGC in bacteria including hyperthermophiles; however, their structures still remain unexplored. Here, we solved the crystal structure of the GGDEF-like domain of Tm0107 protein from Thermotoga maritima at a resolution of 2.1 Å, which shares sequence similarities with DGC proteins in other bacteria. Tm0107 consists of an N-terminal coiled-coil and C-terminal GGDEF-like domain. We showed that the GGDEF-like domain of Tm0107 exists as monomer in solution and is structurally similar to other GGDEF domains. Two zinc ions are coordinated at the interface between two Tm0107 monomers. Based on our measurements of the Stokes radii of Tm0107 by analytical gel filtration, we propose a dimer model of Tm0107 containing both the N-terminal coiled coil and C-terminal GGDEF-like domains. Based on the model, Tm0107 forms a homodimer in a manner different compared to other structurally characterized DGC proteins. These results provide useful structural information about putative DGC proteins containing protein sequences similar to that of Tm0107, which is widely conserved in hyperthermophiles.

Prospective assessment of urinary neutrophil gelatinase-associated lipoprotein in living kidney donors: toward understanding differences between chronic kidney diseases of surgical and medical origin.

OBJECTIVES: To evaluate the clinical implications of postoperative urinary neutrophil gelatinase-associated lipoprotein (NGAL) changes and the association between urinary NGAL (uNGAL) and renal function in living kidney donors. SUBJECTS, PATIENTS AND METHODS: We included 76 healthy adults who underwent donor nephrectomy between December 2013 and November 2014. Perioperative serum creatinine (sCr), uNGAL, serum NGAL (sNGAL), and urinary microalbumin were prospectively measured until 6 months postoperatively. Patients with chronic kidney disease (CKD) due to medical disorders who visited our outpatient clinic during the same period were included for comparison. RESULTS: The mean (SD) preoperative uNGAL of donors was 5 (5.17) ng/mL. uNGAL (corrected for urinary creatinine) was maximal at 1-2 days postoperatively, decreased on postoperative day 3, and stabilised by 7 days after surgery. Postoperative uNGAL was not associated with sex, age, or preoperative renal function. When corrected for sNGAL to compensate for the systemic increase in NGAL with major surgery, uNGAL on days 1-3 postoperatively was negatively correlated with sCr. Postoperatively, donor uNGAL remained higher than preoperatively for up to 6 months but was significantly lower than in patients with medical CKD with similar glomerular filtration rates. CONCLUSION: Acute kidney injury due to hyperfiltration of remnant kidney after donor nephrectomy was maximal within 1-2 days postoperatively. The rise in uNGAL during this period in donors was negatively correlated with postoperative sCr levels. Decreased renal function after nephrectomy differs from that of medical CKD.

Antimicrobial and Immunomodulatory Properties and Applications of Marine-Derived Proteins and Peptides.

Marine organisms provide an abundant source of potential medicines. Many of the marine-derived biomaterials have been shown to act as different mechanisms in immune responses, and in each case they can significantly control the immune system to produce effective reactions. Marine-derived proteins, peptides, and protein hydrolysates exhibit various physiologic functions, such as antimicrobial, anticancer, antioxidant, antihypertensive, and anti-inflammatory activities. Recently, the immunomodulatory properties of several antimicrobial peptides have been demonstrated. Some of these peptides directly kill bacteria and exhibit a variety of immunomodulatory activities that improve the host innate immune response and effectively eliminate infection. The properties of immunomodulatory proteins and peptides correlate with their amino acid composition, sequence, and length. Proteins and peptides with immunomodulatory properties have been tested in vitro and in vivo, and some of them have undergone different clinical and preclinical trials. This review provides a comprehensive overview of marine immunomodulatory proteins, peptides, and protein hydrolysates as well as their production, mechanisms of action, and applications in human therapy.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor.

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

Structural insights into the apo-structure of Cpf1 protein from Francisella novicida.

Clustered regularly interspaced short palindromic repeats (CRISPRs) from Prevotella and Francisella 1 (Cpf1) are RNA-guided endonucleases that produce cohesive double-stranded breaks in DNA by specifically recognizing thymidine-rich protospacer-adjacent motif (PAM) sequences. Cpf1 is emerging as a powerful genome-editing tool. Despite previous structural studies on various Cpf1 proteins, the apo-structure of Cpf1 remains unknown. In the present study, we determined the solution structure of the Cpf1 protein from Francisella novicida (FnCpf1) with and without CRISPR RNA (crRNA) using small-angle X-ray scattering, providing the insights into the apo-structure of FnCpf1. The apo-structure of FnCpf1 was also visualized using negative staining electron microcopy. When we compared the apo-structure of FnCpf1 with crRNA-bound structure, their overall shapes (a closed form) were similar, suggesting that conformational change upon crRNA binding to FnCpf1 is not drastic, but a local induced fit might occur to recognize PAM sequences. In contrast, the apo Cpf1 from Moraxella bovoculi 237 (MbCpf1) was analyzed as an open form, implying that a large conformational change from an open to a closed form might be required for crRNA binding to MbCpf1. These results suggested that the crRNA-induced conformational changes in Cpf1 differ among species.

Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells.

Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55-TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx3Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx3YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx3Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.

Molecular characterization and expression analysis of olive flounder (Paralichthys olivaceus) phospholipase C gamma 1 and gamma 2.

Phospholipase C gamma 1 and gamma 2 (PLCG1 and PLCG2) are influential in modulating Ca2+ and diacylglycerol, second messengers involved in tyrosine kinase-dependent signaling, including growth factor activation. Here, we used RACE (rapid amplification of cDNA ends) to clone cDNA encoding PLCG1 (PoPLCG1) and PLCG2 (PoPLCG2) in the olive flounder (Paralichthys olivaceus). The respective 1313 and 1249 amino acid sequences share high identity with human PLCG1 and PLCG2, and contain the following domains: pleckstrin homology (PH), EF-hand, catalytic X and Y, Src homology 2 (SH2), Src homology 3 (SH3), and C2. Phylogenic analysis and sequence comparison of PoPLCG1 and PoPLCG2 with other PLC isozymes showed a close relationship between the two PLCGs, supported by structural analysis. In addition, tissue expression analysis showed that PoPLCG1 was expressed predominantly in the brain, eye, and heart, whereas PoPLCG2 was expressed principally in gills, esophagus, spleen, and kidney. Following stimulation with LPS and Poly I:C, PoPLCG expression was compared with the expression of inflammatory cytokines IL-1ß, IL-6, and TNF-α via reverse transcription-PCR and real-time quantitative PCR. Our results suggest that PoPLCG isozymes perform a critical immune function in olive flounder, being active in pathogen resistance and the inflammation process.