Deficiency of glucocorticoid receptor in bone marrow adipocytes has mild effects on bone and hematopoiesis but does not influence expansion of marrow adiposity with caloric restriction.

Introduction: Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. Methods: In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Results: Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and µCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4 h into the light cycle. Discussion: In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.

Trends in the multiple prescriptions of hypnotic drugs in a university outpatient in Japan.

AIMS: In Japan, the daily dosage of hypnotic drugs for insomnia treatment is increasing year by year, and over-dependence on treatment with hypnotic drugs is a major problem. This study aimed to examine the factors related to the elimination of prescriptions of three or more hypnotic drugs within 1 year in our clinic. METHODS: We conducted two surveys. Survey ① assessed the frequency of prescriptions of three or more hypnotic drugs by retrospectively reviewing the medical records of all patients who visited general and psychiatric outpatient clinics from January 2013 to March 2019. Survey ② assessed changes in prescriptions of hypnotic and psychotropic drugs within the subsequent year by retrospectively reviewing the medical records of all patients prescribed three or more hypnotic drugs who visited neuropsychiatric outpatient clinics multiple times between April 2013 and March 2019. RESULTS: The frequency of prescribing three or more hypnotic drugs was six to nine times higher in psychiatry than in other departments. Flunitrazepam and brotizolam were the most common drugs prescribed and had the second lowest discontinuation rate after zolpidem. Conversely, eszopiclone, zopiclone, and suvorexant had the highest discontinuation rates. The success factors for drug reduction were age (odds ratio [OR]: 0.97, p < 0.0037), trazodone addition (OR: 12.86, p < 0.0194) and number of years of psychiatric experience. CONCLUSIONS: The characteristics and success factors in relation to drug reduction in patients with multiple prescriptions of hypnotic drugs identified in this study may contribute to solving the problem of multiple prescriptions of hypnotic drugs.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

Constitutive bone marrow adipocytes suppress local bone formation.

BM adipocytes (BMAd) are a unique cell population derived from BM mesenchymal progenitors and marrow adipogenic lineage precursors. Although they have long been considered to be a space filler within bone cavities, recent studies have revealed important physiological roles in hematopoiesis and bone metabolism. To date, the approaches used to study BMAd function have been confounded by contributions by nonmarrow adipocytes or by BM stromal cells. To address this gap in the field, we have developed a BMAd-specific Cre mouse model to deplete BMAds by expression of diphtheria toxin A (DTA) or by deletion of peroxisome proliferator-activated receptor gamma (Pparg). We found that DTA-induced loss of BMAds results in decreased hematopoietic stem and progenitor cell numbers and increased bone mass in BMAd-enriched locations, including the distal tibiae and caudal vertebrae. Elevated bone mass appears to be secondary to enhanced endosteal bone formation, suggesting a local effect caused by depletion of BMAd. Augmented bone formation with BMAd depletion protects mice from bone loss induced by caloric restriction or ovariectomy, and it facilitates the bone-healing process after fracture. Finally, ablation of Pparg also reduces BMAd numbers and largely recapitulates high-bone mass phenotypes observed with DTA-induced BMAd depletion.

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits.

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Suppression of optineurin impairs the progression of hepatocellular carcinoma through regulating mitophagy.

Autophagy removes damaged organelles to inhibit malignant transformation during tumor initiation. Once a cancer matures, it uses the autophagic pathway as an energy source. Optineurin (OPTN) is an autophagy adaptor protein that recruits microtubule-associated protein 1 light chain 3, an autophagosome marker, to the autophagosome. Despite studies of the relation between cancer progression and autophagy adaptor proteins, there are no reports to our knowledge of a correlation between hepatocellular carcinoma (HCC) and OPTN. We aimed here to investigate the effects of OPTN expression on HCC progression through autophagy. Immunohistochemistry was used to measure the OPTN expression in the tissues of 141 Japanese patients with HCC. The effects of OPTN expression on HCC progression and mitophagy were assessed using an OPTN knockout (KO) cell line in vitro. We used this KO cell line to establish and exploit a mouse model of HCC to determine the effects of OPTN expression on tumor progression. Immunohistochemical analysis showed that patients with elevated expression of OPTN experienced shorter overall survival (OS) and recurrence-free survival (RFS). OPTN KO cells proliferated relatively slower versus wild-type (WT) cells in vitro. Western blot analysis showed that mitophagy was suppressed in OPTN KO cells, and ATP synthesis and beta-oxidation were reduced. The mouse model of HCC showed that OPTN KO cells formed smaller tumors versus WT cells less 10 weeks after implantation. Overall, the present findings suggest that OPTN is a key mediator of mitophagy that contributes to HCC progression through mitochondrial energy production.

Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Wnt/ß-catenin signaling regulates adipose tissue lipogenesis and adipocyte-specific loss is rigorously defended by neighboring stromal-vascular cells.

OBJECTIVE: Canonical Wnt/ß-catenin signaling is a well-studied endogenous regulator of mesenchymal cell fate determination, promoting osteoblastogenesis and inhibiting adipogenesis. However, emerging genetic evidence in humans links a number of Wnt pathway members to body fat distribution, obesity, and metabolic dysfunction, suggesting that this pathway also functions in adipocytes. Recent studies in mice have uncovered compelling evidence that the Wnt signaling pathway plays important roles in adipocyte metabolism, particularly under obesogenic conditions. However, complexities in Wnt signaling and differences in experimental models and approaches have thus far limited our understanding of its specific roles in this context. METHODS: To investigate roles of the canonical Wnt pathway in the regulation of adipocyte metabolism, we generated adipocyte-specific ß-catenin (ß-cat) knockout mouse and cultured cell models. We used RNA sequencing, ChIP sequencing, and molecular approaches to assess expression of Wnt targets and lipogenic genes. We then used functional assays to evaluate effects of ß-catenin deficiency on adipocyte metabolism, including lipid and carbohydrate handling. In mice maintained on normal chow and high-fat diets, we assessed the cellular and functional consequences of adipocyte-specific ß-catenin deletion on adipose tissues and systemic metabolism. RESULTS: We report that in adipocytes, the canonical Wnt/ß-catenin pathway regulates de novo lipogenesis (DNL) and fatty acid monounsaturation. Further, ß-catenin mediates effects of Wnt signaling on lipid metabolism in part by transcriptional regulation of Mlxipl and Srebf1. Intriguingly, adipocyte-specific loss of ß-catenin is sensed and defended by CD45-/CD31- stromal cells to maintain tissue-wide Wnt signaling homeostasis in chow-fed mice. With long-term high-fat diet, this compensatory mechanism is overridden, revealing that ß-catenin deletion promotes resistance to diet-induced obesity and adipocyte hypertrophy and subsequent protection from metabolic dysfunction. CONCLUSIONS: Taken together, our studies demonstrate that Wnt signaling in adipocytes is required for lipogenic gene expression, de novo lipogenesis, and lipid desaturation. In addition, adipose tissues rigorously defend Wnt signaling homeostasis under standard nutritional conditions, such that stromal-vascular cells sense and compensate for adipocyte-specific loss. These findings underscore the critical importance of this pathway in adipocyte lipid metabolism and adipose tissue function.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Rimonabant suppresses RNA transcription of hepatitis B virus by inhibiting hepatocyte nuclear factor 4α.

Chronic infection with hepatitis B virus (HBV) sometime induces lethal cirrhosis and hepatocellular carcinoma. Although nucleot(s)ide analogs are used as main treatment for HBV infection, the emergence of the drug-resistant viruses has become a problem. To discover novel antivirals with low side effects and low risk of emergence of resistant viruses, screening for anti-HBV compounds was performed with compound libraries of inhibitors targeting G-protein-coupled receptors (GPCRs). HepG2-hNTCP C4 cells infected with HBV were treated with various GPCR inhibitors and harvested at 14 day postinfection for quantification of core protein in the first screening or relaxed circular DNA in the second screening. Finally, we identified a cannabinoid receptor 1 inhibitor, rimonabant, as a candidate showing anti-HBV effect. In HepG2-hNTCP C4 cells, treatment with rimonabant suppressed HBV propagation at the viral RNA transcription step but had no effect on entry or covalently closed circular DNA level. The values of half maximal inhibitory concentration, half maximal effective concentration, and selectivity index of rimonabant in primary human hepatocyte (PHH) are 2.77 µm, 40.4 µm, and 14.6, respectively. Transcriptome analysis of rimonabant-treated primary hepatocytes by RNA sequencing revealed that the transcriptional activity of hepatocyte nuclear factor 4α (HNF4α), which is known to stimulate viral RNA synthesis, was depressed. By treatment of PHH with rimonabant, the expression level of HNF4α protein and the production of the messenger RNAs (mRNAs) of downstream factors promoted by HNF4α were reduced while the amount of HNF4α mRNA was not altered. These results suggest that treatment with rimonabant suppresses HBV propagation through the inhibition of HNF4α activity.

In Vivo Dynamics of Reporter Flaviviridae Viruses.

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

G-CSF partially mediates effects of sleeve gastrectomy on the bone marrow niche.

Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.

Bone marrow adipocytes resist lipolysis and remodeling in response to ß-adrenergic stimulation.

Bone marrow adipose tissue (BMAT) is preserved or increased in states of caloric restriction. Similarly, we found that BMAT in the tail vertebrae, but not the red marrow in the tibia, resists loss of neutral lipid with acute, 48-hour fasting in rats. The mechanisms underlying this phenomenon and its seemingly distinct regulation from peripheral white adipose tissue (WAT) remain unknown. To test the role of ß-adrenergic stimulation, a major regulator of adipose tissue lipolysis, we examined the responses of BMAT to ß-adrenergic agonists. Relative to inguinal WAT, BMAT had reduced phosphorylation of hormone sensitive lipase (HSL) after treatment with pan-ß-adrenergic agonist isoproterenol. Phosphorylation of HSL in response to ß3-adrenergic agonist CL316,243 was decreased by an additional ~90% (distal tibia BMAT) or could not be detected (tail vertebrae). Ex vivo, adrenergic stimulation of lipolysis in purified BMAT adipocytes was also substantially less than iWAT adipocytes and had site-specific properties. Specifically, regulated bone marrow adipocytes (rBMAs) from proximal tibia and femur underwent lipolysis in response to both CL316,243 and forskolin, while constitutive BMAs from the tail responded only to forskolin. This occurred independently of changes in gene expression of ß-adrenergic receptors, which were similar between adipocytes from iWAT and BMAT, and could not be explained by defective coupling of ß-adrenergic receptors to lipolytic machinery through caveolin 1. Specifically, we found that whereas caveolin 1 was necessary to mediate maximal stimulation of lipolysis in iWAT, overexpression of caveolin 1 was insufficient to rescue impaired BMAT signaling. Lastly, we tested the ability of BMAT to respond to 72-hour treatment with CL316,243 in vivo. This was sufficient to cause beiging of iWAT adipocytes and a decrease in iWAT adipocyte cell size. By contrast, adipocyte size in the tail BMAT and distal tibia remained unchanged. However, within the distal femur, we identified a subpopulation of BMAT adipocytes that underwent lipid droplet remodeling. This response was more pronounced in females than in males and resembled lipolysis-induced lipid partitioning rather than traditional beiging. In summary, BMAT has the capacity to respond to ß-adrenergic stimuli, however, its responses are muted and BMAT generally resists lipid hydrolysis and remodeling relative to iWAT. This resistance is more pronounced in distal regions of the skeleton where the BMAT adipocytes are larger with little intervening hematopoiesis, suggesting that there may be a role for both cell-autonomous and microenvironmental determinants. Resistance to ß-adrenergic stimuli further separates BMAT from known regulators of energy partitioning and contributes to our understanding of why BMAT is preserved in states of fasting and caloric restriction.

Monitoring substrate enables real-time regulation of a protein localization pathway.

Protein localization machinery supports cell survival and physiology, suggesting the potential importance of its expression regulation. Here, we summarize a remarkable scheme of regulation, which allows real-time feedback regulation of the machinery expression. A class of regulatory nascent polypeptides, called monitoring substrates, undergoes force-sensitive translation arrest. The resulting ribosome stalling on the mRNA then affects mRNA folding to expose the ribosome-binding site of the downstream target gene and upregulate its translation. The target gene encodes a component of the localization machinery, whose physical action against the monitoring substrate leads to arrest cancellation. Thus, this scheme of feedback loop allows the cell to adjust the amount of the machinery to correlate inversely with the effectiveness of the process at a given moment. The system appears to have emerged late in evolution, in which a narrow range of organisms selected a distinct monitoring substrate-machinery combination. Currently, regulatory systems of SecM-SecA, VemP-SecDF2 and MifM-YidC2 are known to occur in different bacterial species.

Late diagnosis: a case of rapidly progressive extranodal NK/T cell lymphoma, nasal type.

Extranodal natural killer (NK)/T cell lymphoma, nasal type is a condition that has poor prognosis. Accurate diagnosis of lymphoma is made by pathological findings. We report a case of extranodal NK/T cell lymphoma, nasal type affecting the lung and liver and which was difficult to diagnose because of negative biopsy results from multiple sites. A 39-year-old man who had dry cough and fever for 1 month was referred to our hospital. He had pancytopenia and elevated serum levels of lactate dehydrogenase and soluble interleukin-2 receptor. Hepatosplenomegaly and multiple lung nodules were found on imaging study. Specimens of bronchoscopic lung, percutaneous liver, bone marrow and random skin biopsies were all negative. Open lung biopsy was not definitive. Unfortunately, disease progression was rapid and fatal before results of pleural fluid cytology and a second liver biopsy showed extranodal NK/T cell lymphoma, nasal type. This report focused on diagnostic planning for rapidly progressive extranodal NK/T-cell lymphoma, nasal type.

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.

VemP ( Vibrio protein export monitoring polypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156 codon of vemP encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met120-Phe137) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.

Iron elevation and adipose tissue remodeling in the epididymal depot of a mouse model of polygenic obesity.

BACKGROUND: Iron dysregulation is a potential contributor to the pathology of obesity-related metabolic complications. KK/HIJ (KK) mice, a polygenic obese mouse model, have elevated serum iron levels. A subset of KK male mice display a bronzing of epididymal adipose tissue (eAT) associated with >100-fold (p<0.001) higher iron concentration. METHODS: To further phenotype and characterize the adipose tissue iron overload, 27 male KK mice were evaluated. 14 had bronzing eAT and 13 had normal appearing eAT. Fasting serum and tissues were collected for iron content, qPCR, histology and western blot. RESULTS: High iron levels were confirmed in bronzing eAT (High Iron group, HI) versus normal iron level (NI) in normal appearing eAT. Surprisingly, iron levels in subcutaneous and brown adipose depots were not different between the groups (p>0.05). The eAT histology revealed iron retention, macrophage clustering, tissue fibrosis, cell death as well as accumulation of HIF-2α in the high iron eAT. qPCR showed significantly decreased Lep (leptin) and AdipoQ (adiponectin), whereas Tnfα (tumor necrosis factor α), and Slc40a1 (ferroportin) were up-regulated in HI (p<0.05). Elevated HIF-2α, oxidative stress and local insulin signaling loss was also observed. SIGNIFICANCE: Our data suggest that deposition of iron in adipose tissue is limited to the epididymal depot in male KK mice. A robust adipose tissue remodeling is concomitant with the high iron concentration, which causes local adipose tissue insulin resistance.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae.

Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.

Tunnel Formation Inferred from the I-Form Structures of the Proton-Driven Protein Secretion Motor SecDF.

Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.