Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.

The Pro-Oxidant Activity of Pheomelanin is Significantly Enhanced by UVA Irradiation: Benzothiazole Moieties Are More Reactive than Benzothiazine Moieties.

It is generally considered that eumelanin (EM) is photoprotective while pheomelanin (PM) is phototoxic. A recent study using a mouse model demonstrated that PM produces reactive oxygen species (ROS) that cause DNA damage and eventually lead to melanomagenesis. A biochemical study showed that PM possesses a pro-oxidant activity. PM consists of benzothiazine (BT) and benzothiazole (BZ) moieties, BT moieties being transformed to BZ moieties by heat or light. In this study, we compared the effects of ultraviolet A (UVA) irradiation using synthetic PMs with different BT to BZ ratios and using various coat color mouse hairs. We found that UVA irradiation of BZ-PM increased glutathione (GSH) depletion and generated more H2O2 than UVA irradiation of BT-PM. Non-irradiated controls did not exhibit strong pro-oxidant activities. Upon UVA irradiation, yellow mouse hairs oxidized GSH and produced H2O2 faster than black or albino mouse hairs. Next, to examine the mechanism of the pro-oxidant activity of BT-PM and BZ-PM, we examined the pro-oxidant activities of 7-(2-amino-2-carboxyethyl)-dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) and 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole (BZ-AA) as BT and BZ monomers, respectively. Their pro-oxidant activities were similar, but a large difference was seen in the effects of ROS scavengers, which suggests that the redox reactions may proceed via singlet oxygen in BZ-AA and via superoxide anions in DHBTCA. These results show that UVA enhances the pro-oxidant activity of PM, in particular BZ-PM.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Effects of growth hormone on thyroid function are mediated by type 2 iodothyronine deiodinase in humans.

PURPOSE: Growth hormone (GH) therapy in adults alters thyroid function, and acromegaly often involves thyroid disease. The present study aimed to elucidate roles and mechanisms of GH in regulating thyroid function. METHODS: We performed two retrospective observational studies, which focused on consecutive patients with severe adult GH deficiency who received recombinant human GH (rhGH) therapy (n = 20) and consecutive patients with acromegaly who underwent transsphenoidal surgery (TSS) (n = 25). In both studies, serum free triiodothyronine (fT3), free thyroxine (fT4), and fT3/fT4 ratio were examined before and after the interventions. We subsequently administered GH to four human cell lines (HepG2, TSA201, MCF7, and HTC/C3) in vitro, and examined changes in mRNA levels of iodothyronine deiodinases (D1, D2, and D3). RESULTS: Median serum fT3 level significantly increased after rhGH therapy from 2.38 to 2.78 pg/mL (p < 0.001), and fT4 decreased from 1.115 to 1.065 ng/dL (p = 0.081). TSS significantly decreased median serum fT3 from 3.03 to 2.53 pg/mL (p < 0.001), and increased fT4 from 1.230 to 1.370 ng/dL (p < 0.001). In vitro, GH significantly increased D2 expression at the mRNA level in HTC/C3 cells (p < 0.01), as well as D2 protein and its activity. CONCLUSIONS: GH increased serum fT3 level and decreased serum fT4 level in humans. Our results suggest that its mechanism involves D2 upregulation. Considering this GH effect on thyroid hormone metabolism, data on thyroid function could be useful in the management of GH deficiency and acromegaly.

Targeted Disruption of JCAD (Junctional Protein Associated With Coronary Artery Disease)/KIAA1462, a Coronary Artery Disease-Associated Gene Product, Inhibits Angiogenic Processes In Vitro and In Vivo.

OBJECTIVE: Recent genome-wide association studies newly identified the human KIAA1462 gene as a new locus for coronary artery disease. However, the function of the gene product, named JCAD (junctional protein associated with coronary artery disease), is unknown. Because JCAD is expressed at cell-cell junctions in endothelial cells, we hypothesized and tested whether JCAD regulates angiogenic processes in vitro and in vivo. APPROACH AND RESULTS: Cell culture experiments revealed impaired angiogenic ability (proliferation, migration, and cord formation) by the knockdown of JCAD with siRNA (P<0.05 versus control siRNA). We have generated mice lacking JCAD (mKIAA1462-/-) by gene-targeted deletion of JCAD to address in vivo angiogenic function. mKIAA1462-/- mice did not show morphological differences in development of retinal vasculature. Ex vivo aortic ring model demonstrated impaired neovascularization in aorta from mKIAA1462-/- mice than control wild-type mice (P<0.05). Tumor growth was assessed by monitoring tumor volume after the subcutaneous injection of melanoma, LLC (Lewis lung carcinoma), and E0771 cells into the mice. mKIAA1462-/- mice exhibited significantly smaller tumor volume compared with wild-type mice (P<0.001). Histological assessment of the tumor exhibited less smooth muscle actin-positive neovascularization determined by CD31-positive vascular structure in tumor of mKIAA1462-/- mice than wild-type mice, indicating that knockdown of JCAD inhibited the vascular maturation in pathological angiogenic process. CONCLUSIONS: These in vitro and in vivo studies suggest that JCAD has a redundant functional role in physiological angiogenesis but serves a pivotal role in pathological angiogenic process after birth.

Clinical Features of Nivolumab-Induced Thyroiditis: A Case Series Study.

BACKGROUND: The programmed cell death-1 (PD-1) pathway is a novel therapeutic target in immune checkpoint therapy for cancer. It consists of the PD-1 receptor and its two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). Nivolumab is an anti-PD-1 monoclonal antibody approved for malignant melanoma, advanced non-small cell lung cancer, and advanced renal cell carcinoma in Japan. Thyrotoxicosis and hypothyroidism have both been reported in international Phase 3 studies and national post-marketing surveillance of nivolumab in Japan. METHODS: This study analyzed five consecutive cases with thyroid dysfunction associated with nivolumab therapy. Second, it examined the mRNA and protein expressions of PD-L1 and PD-L2 by reverse transcription polymerase chain reaction and Western blotting. RESULTS: All patients were diagnosed with painless thyroiditis. Thyrotoxicosis developed within four weeks from the first administration of nivolumab and normalized within four weeks of onset in three of the five patients. Hypothyroidism after transient thyrotoxicosis developed in two patients, and preexisting hypothyroidism persisted in one patient. The other two patients were treated with glucocorticoids and discontinued nivolumab therapy for comorbid adverse events. One did not develop hypothyroidism, and the other developed mild, transient hypothyroidism. In addition, it was verified that normal thyroid tissue expresses PD-L1 and PD-L2 mRNA and those proteins. CONCLUSIONS: In the present cases, nivolumab-induced thyrotoxicosis seemed to be associated with painless thyroiditis, while no patient with Graves' disease was observed. A transient and rapid course with subsequent hypothyroidism was observed in nivolumab-induced thyroiditis. In addition, it was verified that PD-L1 and PD-L2 are expressed in normal thyroid tissue. This suggests that nivolumab therapy reduces immune tolerance, even in normal thyroid tissue, and leads to the development of thyroiditis. Treating thyrotoxicosis with only supportive care and considering levothyroxine replacement therapy once subsequent hypothyroidism occurs is proposed. Further investigations are required to confirm whether glucocorticoid therapy and discontinuation of nivolumab therapy prevent subsequent hypothyroidism.

Sucrose sensing through nascent peptide-meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2.

Arabidopsis bZIP11 is a transcription factor that modulates amino acid metabolism under high-sucrose conditions. Expression of bZIP11 is downregulated in a sucrose-dependent manner during translation. Previous in vivo studies have identified the second upstream open reading frame (uORF2) as an essential regulatory element for the sucrose-dependent translational repression of bZIP11. However, it remains unclear how uORF2 represses bZIP11 expression under high-sucrose conditions. Through biochemical experiments using cell-free translation systems, we report on sucrose-mediated ribosome stalling at the stop codon of uORF2. The C-terminal 10 amino acids (29-SFSVxFLxxLYYV-41) of uORF2 are important for ribosome stalling. Our results demonstrate that uORF2 encodes a regulatory nascent peptide that functions to sense intracellular sucrose abundance. This is the first biochemical identification of the intracellular sucrose sensor.

Rapid exacerbation of lymphocytic infundibuloneurohypophysitis.

RATIONALE: Lymphocytic hypophysitis is a relatively rare autoimmune disease defined by lymphocytic infiltration to the pituitary. Its rarity and wide spectrum of clinical manifestations make clarification of the pathology difficult. Here, we describe a case we examined from the primary diagnosis to final discharge, showing the serial progression of lymphocytic infundibuloneurohypophysitis (LINH) to panhypopituitarism with extrapituitary inflammatory invasion in a short period, and responding favorably to high-dose glucocorticoid treatment. PATIENT CONCERNS: Polyuria, General fatigue and Nausea/Vomiting. DIAGNOSES: Central diabetes insipidus (CDI), Lymphocytic infundibuloneurohypophysitis (LINH). INTERVENTIONS: Desmopressin acetate, High-dose glucocorticoid (GC) treatment. OUTCOMES: He was prescribed desmopressin acetate and subsequently discharged. A month later, he revisited our hospital with general fatigue and nausea/vomiting. A screening test disclosed hypopituitarism with adrenal insufficiency. MRI revealed expanded contrast enhancement to the peripheral extrapituitary lesion. He received high-dose GC treatment and the affected lesion exhibited marked improvement on MRI, along with the recovery of the anterior pituitary function. LESSONS: This case demonstrates the potential for classical LINH to develop into panhypopituitarsim. We consider this is the first documentation of approaching the cause of atypical LINH with progressive clinical course from the pathological viewpoint.

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.

The Transcriptional Modulator Interferon-Related Developmental Regulator 1 in Osteoblasts Suppresses Bone Formation and Promotes Bone Resorption.

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-κB, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-κB-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between ß-catenin and HDAC, subsequently increasing the acetylation of ß-catenin at K49, leading to its nuclear accumulation and the activation of the ß-catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-κB/Smad/Osx and ß-catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases.

The N-terminal cleavable pre-sequence encoded in the first exon of cystathionine γ-synthase contains two different functional domains for chloroplast targeting and regulation of gene expression.

Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine γ-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1:GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1:GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.

Ribosomes in a stacked array: elucidation of the step in translation elongation at which they are stalled during S-adenosyl-L-methionine-induced translation arrest of CGS1 mRNA.

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

A halt in poly(A) shortening during S-adenosyl-L-methionine-induced translation arrest in CGS1 mRNA of Arabidopsis thaliana.

Cystathionine γ-synthase (CGS) catalyzes the first committed step of methionine (Met) biosynthesis in plants. Expression of the Arabidopsis thaliana CGS1 gene is negatively feedback-regulated in response to the direct Met metabolite S-adenosyl-L-methionine (AdoMet). This regulation occurs at the step of mRNA stability during translation and is coupled with AdoMet-induced CGS1-specific translation arrest. In general, mRNA decay is initiated by a shortening of the poly(A) tail. However, this process has not been studied in detail in cases where regulatory events, such as programmed translation arrest, are involved. Here, we report that the poly(A) tail of the full-length CGS1 mRNA showed an apparent increase from 50-80 nucleotides (nt) to 140-150 nt after the induction of CGS1 mRNA degradation. This finding was unexpected because mRNAs that are destined for degradation harbored longer poly(A) tail than mRNAs that were not targeted for degradation. The results suggest that poly(A) shortening is inhibited or delayed during AdoMet-induced translation arrest of CGS1 mRNA. We propose an explanation for this phenomenon that remains consistent with the recent model of actively translating mRNA. We also found that CGS1 mRNA degradation intermediates, which are 5'-truncated forms of CGS1 mRNA, had a short poly(A) tail of 10-30 nt. This suggests that poly(A) shortening occurs rapidly on the degradation intermediates. The present study highlights CGS1 mRNA degradation as a useful system to understand the dynamic features of poly(A) shortening.

A case of myelolipoma with bilateral adrenal hyperaldosteronism cured after unilateral adrenalectomy.

Myelolipomas are adrenal tumors composed of both adipose and hematopoietic tissues which are rarely associated with primary aldosteronism (PA). Here, we report a case of myelolipoma associated with PA. Aldosterone hypersecretion from bilateral adrenal glands had been confirmed by adrenal venous sampling and pathological analyses, but PA was clinically cured after surgical removal of the unilateral adrenal gland together with the myelolipoma that was not producing aldosterone. It is suggested that myelolipomas may release some factors which stimulate aldosterone production in adrenal glands, although further investigation is necessary. Obesity-related hyperaldosteronism might in part participate in generation of hypertension in the present case.

Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages.

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-α and interleukin-1ß, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.

S-adenosyl-L-methionine induces compaction of nascent peptide chain inside the ribosomal exit tunnel upon translation arrest in the Arabidopsis CGS1 gene.

Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-L-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.