The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass

Antimicrobial Peptide Evolution in the Asiatic Honey Bee Apis cerana

The Asiatic honeybee, Apis cerana Fabricius, is an important honeybee species in Asian countries. It is still found in the wild, but is also one of the few bee species that can be domesticated. It has acquired some genetic advantages and significantly different biological characteristics compared with other Apis species. However, it has been less studied, and over the past two decades, has become a threatened species in China. We designed primers for the sequences of the four antimicrobial peptide cDNA gene families (abaecin, defensin, apidaecin, and hymenoptaecin) of the Western honeybee, Apis mellifera L. and identified all the antimicrobial peptide cDNA genes in the Asiatic honeybee for the first time. All the sequences were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). In all, 29 different defensin cDNA genes coding 7 different defensin peptides, 11 different abaecin cDNA genes coding 2 different abaecin peptides, 13 different apidaecin cDNA genes coding 4 apidaecin peptides and 34 different hymenoptaecin cDNA genes coding 13 different hymenoptaecin peptides were cloned and identified from the Asiatic honeybee adult workers. Detailed comparison of these four antimicrobial peptide gene families with those of the Western honeybee revealed that there are many similarities in the quantity and amino acid components of peptides in the abaecin, defensin and apidaecin families, while many more hymenoptaecin peptides are found in the Asiatic honeybee than those in the Western honeybee (13 versus 1). The results indicated that the Asiatic honeybee adult generated more variable antimicrobial peptides, especially hymenoptaecin peptides than the Western honeybee when stimulated by pathogens or injury. This suggests that, compared to the Western honeybee that has a longer history of domestication, selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM+/−) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM+/− mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/− mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM

Efficacy and safety of immune checkpoint inhibitor rechallenge in individuals with hepatocellular carcinoma

BACKGROUND & AIMS: We investigated the efficacy and safety of immune checkpoint inhibitor (ICI) rechallenge in patients with hepatocellular carcinoma (HCC) who received ICI-based therapies in a previous systemic line. METHODS: In this international, retrospective multicenter study, patients with HCC who received at least two lines of ICI-based therapies (ICI-1, ICI-2) at 14 institutions were eligible. The main outcomes included best overall response and treatment-related adverse events. RESULTS: Of 994 ICI-treated patients screened, a total of 58 patients (male, n = 41; 71%) with a mean age of 65.0±9.0 years were included. Median systemic treatment lines of ICI-1 and ICI-2 were 1 (range, 1-4) and 3 (range, 2-9), respectively. ICI-based therapies used at ICI-1 and ICI-2 included ICI alone (ICI-1, n = 26, 45%; ICI-2, n = 4, 7%), dual ICI regimens (n = 1, 2%; n = 12, 21%), or ICI combined with targeted therapies/anti-VEGF (n = 31, 53%; n = 42, 72%). Most patients discontinued ICI-1 due to progression (n = 52, 90%). Objective response rate was 22% at ICI-1 and 26% at ICI-2. Responses at ICI-2 were also seen in patients who had progressive disease as best overall response at ICI-1 (n = 11/21; 52%). Median time-to-progression at ICI-1 and ICI-2 was 5.4 (95% CI 3.0-7.7) months and 5.2 (95% CI 3.3-7.0) months, respectively. Treatment-related adverse events of grade 3-4 at ICI-1 and ICI-2 were observed in 9 (16%) and 10 (17%) patients, respectively. CONCLUSIONS: ICI rechallenge was safe and resulted in a treatment benefit in a meaningful proportion of patients with HCC. These data provide a rationale for investigating ICI-based regimens in patients who progressed on first-line immunotherapy in prospective trials. IMPACT AND IMPLICATIONS: Therapeutic sequencing after first-line immune checkpoint inhibitor (ICI)-based therapy for advanced hepatocellular carcinoma (HCC) remains a challenge as no available second-line treatment options have been studied in immunotherapy-pretreated patients. Particularly, the role of ICI rechallenge in patients with HCC is unclear, as data from prospective trials are lacking. We investigated the efficacy and safety of ICI-based regimens in patients with HCC pretreated with immunotherapy in a retrospective, international, multicenter study. Our data provide the rationale for prospective trials investigating the role of ICI-based regimens in patients who have progressed on first-line immunotherapy

Molecular Interpretation of ACTH-β-Endorphin Coaggregation: Relevance to Secretory Granule Biogenesis

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system

POMC: The Physiological Power of Hormone Processing.

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon

Efficacy and safety of immune checkpoint inhibitor rechallenge in individuals with hepatocellular carcinoma

BACKGROUND & AIMS: We investigated the efficacy and safety of immune checkpoint inhibitor (ICI) rechallenge in patients with hepatocellular carcinoma (HCC) who received ICI-based therapies in a previous systemic line. METHODS: In this international, retrospective multicenter study, patients with HCC who received at least two lines of ICI-based therapies (ICI-1, ICI-2) at 14 institutions were eligible. The main outcomes included best overall response and treatment-related adverse events. RESULTS: Of 994 ICI-treated patients screened, a total of 58 patients (male, n = 41; 71%) with a mean age of 65.0±9.0 years were included. Median systemic treatment lines of ICI-1 and ICI-2 were 1 (range, 1-4) and 3 (range, 2-9), respectively. ICI-based therapies used at ICI-1 and ICI-2 included ICI alone (ICI-1, n = 26, 45%; ICI-2, n = 4, 7%), dual ICI regimens (n = 1, 2%; n = 12, 21%), or ICI combined with targeted therapies/anti-VEGF (n = 31, 53%; n = 42, 72%). Most patients discontinued ICI-1 due to progression (n = 52, 90%). Objective response rate was 22% at ICI-1 and 26% at ICI-2. Responses at ICI-2 were also seen in patients who had progressive disease as best overall response at ICI-1 (n = 11/21; 52%). Median time-to-progression at ICI-1 and ICI-2 was 5.4 (95% CI 3.0-7.7) months and 5.2 (95% CI 3.3-7.0) months, respectively. Treatment-related adverse events of grade 3-4 at ICI-1 and ICI-2 were observed in 9 (16%) and 10 (17%) patients, respectively. CONCLUSIONS: ICI rechallenge was safe and resulted in a treatment benefit in a meaningful proportion of patients with HCC. These data provide a rationale for investigating ICI-based regimens in patients who progressed on first-line immunotherapy in prospective trials. IMPACT AND IMPLICATIONS: Therapeutic sequencing after first-line immune checkpoint inhibitor (ICI)-based therapy for advanced hepatocellular carcinoma (HCC) remains a challenge as no available second-line treatment options have been studied in immunotherapy-pretreated patients. Particularly, the role of ICI rechallenge in patients with HCC is unclear, as data from prospective trials are lacking. We investigated the efficacy and safety of ICI-based regimens in patients with HCC pretreated with immunotherapy in a retrospective, international, multicenter study. Our data provide the rationale for prospective trials investigating the role of ICI-based regimens in patients who have progressed on first-line immunotherapy

Characterization of the Conus bullatus genome and its venom-duct transcriptome

<p>Abstract</p> <p>Background</p> <p>The venomous marine gastropods, cone snails (genus <it>Conus</it>), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. <it>Conus bullatus </it>belongs to a clade of <it>Conus </it>species called <it>Textilia</it>, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic <it>Textilia </it>clade.</p> <p>Results</p> <p>We have carried out a sequencing survey of the <it>Conus bullatus </it>genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an <it>in silico </it>pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities.</p> <p>Conclusions</p> <p>Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of <it>Conus bullatus </it>venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of <it>C. bullatus </it>transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.</p