Membrane perturbing factor in reticulocyte lysate, which is transiently activated by proteases

AbstractProteases have been used to examine the topology of proteins on various membranes. We reexamined the conditions of protease treatment for rough microsomal membranes and found that proteinase K degraded the lumenal proteins in the presence of reticulocyte lysate. The lysate treated with either heat or N-ethylmaleimide no longer promoted the degradation. The reticulocyte dependent degradation was also observed with papain, trypsin, and elastase. This activity was transiently generated by treating reticulocyte lysate short-term with trypsin. We thus concluded that a membrane perturbing factor(s) must exist in reticulocyte which is transiently activated by protease treatment

Genetic Interactions Due to Constitutive and Inducible Gene Regulation Mediated by the Unfolded Protein Response in C. elegans

The unfolded protein response (UPR) is an adaptive signaling pathway utilized to sense and alleviate the stress of protein folding in the endoplasmic reticulum (ER). In mammals, the UPR is mediated through three proximal sensors PERK/PEK, IRE1, and ATF6. PERK/PEK is a protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 to inhibit protein synthesis. Activation of IRE1 induces splicing of XBP1 mRNA to produce a potent transcription factor. ATF6 is a transmembrane transcription factor that is activated by cleavage upon ER stress. We show that in Caenorhabditis elegans, deletion of either ire-1 or xbp-1 is synthetically lethal with deletion of either atf-6 or pek-1, both producing a developmental arrest at larval stage 2. Therefore, in C. elegans, atf-6 acts synergistically with pek-1 to complement the developmental requirement for ire-1 and xbp-1. Microarray analysis identified inducible UPR (i-UPR) genes, as well as numerous constitutive UPR (c-UPR) genes that require the ER stress transducers during normal development. Although ire-1 and xbp-1 together regulate transcription of most i-UPR genes, they are each required for expression of nonoverlapping sets of c-UPR genes, suggesting that they have distinct functions. Intriguingly, C. elegans atf-6 regulates few i-UPR genes following ER stress, but is required for the expression of many c-UPR genes, indicating its importance during development and homeostasis. In contrast, pek-1 is required for induction of approximately 23% of i-UPR genes but is dispensable for the c-UPR. As pek-1 and atf-6 mainly act through sets of nonoverlapping targets that are different from ire-1 and xbp-1 targets, at least two coordinated responses are required to alleviate ER stress by distinct mechanisms. Finally, our array study identified the liver-specific transcription factor CREBh as a novel UPR gene conserved during metazoan evolution

A Case of Pulmonary Sarcoma with Significant Extension into the Right Lung

A female patient in her 30s was referred to us with a mass approximately 8 centimeters in diameter in right lung segment 6. Bronchoscopy was done, and a tumorous lesion obstructing right B6 was found. Biopsy of this lesion supported suspicions of sarcoma or spindle cell carcinoma. Contrast-enhanced CT showed that the mass extended to and obstructed the right main pulmonary artery. A skip lesion was also suspected in the periphery of pulmonary artery trunk. The tumor was removed by right pneumonectomy accompanied by resection of the main and left pulmonary arteries under cardiopulmonary bypass. The pulmonary artery trunk and the left pulmonary artery were reconstructed with a vascular graft. Collectively, intimal sarcoma originating from the right main pulmonary artery with extension into the right lung was diagnosed. Significant extension of pulmonary artery sarcoma into the lung, as was observed in the present case, is considered to be rare, and to our knowledge this is the first report in which the primary lesion was biopsied by bronchoscopy

CREBh Is a Novel UPR-Responsive Gene

<div><p>(A) Microarray and quantitative RT-PCR analyses show that the expression of <i>C. elegans</i> (ce) CREBh requires <i>ire-1, xbp-1,</i> and <i>atf-6</i>. “Tuni” indicates tunicamycin treatment, as described in the Materials and Methods section.</p><p>(B) ER stress induced by dithiothreitol in HepG2 cells activates CREBh transcription. HepG2 cells were treated with dithiothreitol and harvested at various time points from 0 h to 8 h. The relative expression of CREBh and spliced <i>xbp-1</i> transcripts (Xbp1s) was analyzed by quantitative RT-PCR and normalized to GADPH. The induction pattern of CREBh resembles that of spliced <i>xbp-1</i> transcripts.</p></div

Complex UPR Transcriptional Regulation of Genes with Known Functions in <i>C. elegans</i>

<div><p>(A) The i-UPR pathway. Many conditions such as exogenous drug treatment, nutrient deprivation, viral infection, or protein overexpression block or overwhelm protein-folding reactions in the ER and result in ER stress. In this study, we used tunicamycin to block protein folding so as to activate the UPR. Following ER stress, <i>ire-1</i> and <i>xbp-1</i> act in a linear pathway that dominates the transcriptional response (total of 139 target genes with known functions), inducing genes that reshape the secretory pathway, adjust the metabolic profile, up-regulate functions involved in calcium homeostasis and anti-oxidative stress, and regulate other genes that might affect cell fate. Interestingly, ten genes require either <i>ire-1</i> or <i>xbp-1,</i> but not both, for their induction upon ER stress. About 21 genes require only <i>pek-1</i> for maximal induction, and eight genes regulated by <i>ire-1/xbp-1</i> also share regulation by <i>pek-1</i>. Finally, <i>atf-6</i> does not play a significant role in the i-UPR pathway (depicted by broken arrow). In addition to two genes that were also regulated by <i>ire-1/xbp-1,</i> the only gene that depends solely on <i>atf-6</i> for its induction is <i>cht-1,</i> which encodes a chitinase orthologous to human chitotriosidase.</p><p>(B) The c-UPR pathway. During development, active protein synthesis and secretion require the UPR signaling molecules <i>ire-1, xbp-1,</i> and <i>atf-6</i> to maintain the expression of c-UPR genes, defined by the fact that they are not up-regulated by tunicamycin but are dependent on ER stress transducers for expression. Among the genes with known functions, there are only 12 that overlap between the set of 45 genes regulated by <i>ire-1</i> and the set of 160 genes regulated by <i>xbp-1</i>. In addition, <i>atf-6</i> is required by nine genes that are regulated by <i>ire-1</i> and 35 that are genes regulated by <i>xbp-1</i>. Moreover, the expression of 19 genes is solely dependent on <i>atf-6,</i> suggesting an important role of <i>atf-6</i> in the c-UPR pathway. By contrast, <i>pek-1</i> is largely dispensable for regulation of the c-UPR as only nine genes require <i>pek-1</i> in addition to their requirements for <i>ire-1</i> to maintain basal expression.</p></div

Transcriptional Targets of <i>ire-1</i>, <i>xbp-1</i>, <i>pek-1,</i> and <i>atf-6</i>

<div><p>(A) Venn diagram showing the sets of i-UPR genes regulated by <i>ire-1, xbp-1, pek-1,</i> and <i>atf-6</i>.</p><p>(B) Venn diagram showing the sets of c-UPR genes regulated by <i>ire-1, xbp-1, pek-1,</i> and <i>atf-6.</i></p></div

<i>C. elegans atf-6</i> and <i>pek-1</i> Display Partially Redundant Roles in Complementing <i>ire-1/xbp-1</i> for Larval Survival and Development

<div><p>(A) Characterization of <i>C. elegans atf-6</i> and its <i>ok551</i> deletion allele. The <i>atf-6</i> gene structure is depicted in boxes and lines, representing exons and introns, respectively. The <i>atf-6(ok551)</i> allele lacks 1,900 bp of genomic sequence, and has the potential to encode a protein without the leucine zipper portion of the bZIP domain, the transmembrane domain, and ER lumenal domain. The <i>atf-6(ok551)</i> deletion allele can be detected by PCR, using the primers indicated by arrows.</p><p>(B) Genetic interactions of <i>atf-6, ire-1,</i> and <i>pek-1</i>. Animals with the genotype <i>ire-1(v33); atf-6(ok551)</i> arrested as young larvae, showing that loss of both <i>ire-1</i> and <i>atf-6</i> is lethal. The <i>ire-1(v33)/ mnC1; atf-6(ok551)/ pek-1(ok275)</i> animals (P0) segregated healthy F1 progeny with the genotype <i>ire-1(v33); atf-6(ok551)/ pek-1(ok275),</i> which in turn produced dead F2 animals with exactly the same genotype, suggesting that ATF-6 and PEK-1 function synergistically to cope with endogenous ER stress during development.</p><p>(C) Nomarski micrograph of a 3-d-old <i>atf-6(RNAi); ire-1(v33)</i> animal. The germline of this animal did not develop past the L2 larval stage.</p><p>(D) Comparisons of intestinal degeneration in various double mutants: (i) <i>ire-1(v33); pek-1(ok275),</i> (ii) <i>xbp-1(RNAi); pek-1(ok275),</i> (iii) <i>ire-1(v33); atf-6(RNAi),</i> and (iv) <i>atf-6(RNAi); pek-1(ok275</i>). Normaski micrographs show a portion of the intestine. Mutants in (i)–(iii) arrested at the L2 larval stage and showed intestinal degeneration. The mutants in (iv) had an intestinal morphology similar to the wild-type. Yellow arrows indicate vacuoles in intestinal cells. Red arrowheads indicate light-reflective aggregates appearing in some mutants ([ii]and [iii]).</p></div