Goll, Darrel E., Valery F. Thompson, Hongqi Li, Wei Wei, and Jinyang Cong. The Calpain System. Physiol Rev 83: 731–801, 2003; 10.1152/physrev.00029.2002.—The calpain system originally comprised three molecules: two Ca2+-dependent proteases, μ-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both μ- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55–65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six “domains” in the 80-kDa subunit: 1) a 19-amino acid NH2-terminal sequence; 2) and 3) two domains that constitute the active site, IIa and IIb; 4) domain III; 5) an 18-amino acid extended sequence linking domain III to domain IV; and 6) domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of μ- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.
A family of mesoporous nonprecious metal (NPM) catalysts for oxygen reduction reaction (ORR) in acidic media, including cobalt−nitrogen-doped carbon (C−N−Co) and iron−nitrogen-doped carbon (C−N− Fe), was prepared from vitamin B12 (VB12) and the polyaniline-Fe (PANI-Fe) complex, respectively. Silica nanoparticles, ordered mesoporous silica SBA-15, and montmorillonite were used as templates for achieving mesoporous structures. The most active mesoporous catalyst was fabricated from VB12 and silica nanoparticles and exhibited a remarkable ORR activity in acidic medium (half-wave potential of 0.79 V, only ∼58 mV deviation from Pt/C), high selectivity (electron-transfer number >3.95), and excellent electrochemical stability (only 9 mV negative shift of half-wave potential after 10 000 potential cycles). The unprecedented performance of these NPM catalysts in ORR was attributed to their well-defined porous structures with a narrow mesopore size distribution, high Brunauer−Emmett−Teller surface area (up to 572 m 2 /g), and homogeneous distribution of abundant metal−N x active sites.
The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF−/−) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF−/− mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.
Cancer cells survive cellular crisis through telomere maintenance mechanisms. We report telomere lengths in 18,430 samples, including tumors and non-neoplastic samples, across 31 cancer types. Tumor telomeres were shorter compared to normal tissues, and longer in sarcomas and gliomas compared to other cancers. Amongst 6,835 cancers, 73% expressed telomerase reverse transcriptase (TERT), which was associated with TERT point mutations, rearrangements, DNA amplifications, and transcript fusions, and predicted telomerase activity. TERT promoter methylation provided an additional deregulatory TERT expression mechanism. Five percent of cases, mostly with undetectable TERT, harbored ATRX or DAXX alterations, demonstrated elongated telomeres and increased telomeric repeat containing RNA (TERRA). The remaining 22% of tumors neither expressed TERT, nor harbored alterations in ATRX/DAXX. In this group, telomere length positively correlated with TP53 and RB1 mutations. Our analysis integrates TERT abnormalities, telomerase activity and genomic alterations with telomere length in cancer.
Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ϳ400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.Retrotransposons are DNA sequences that can move (i.e., retrotranspose) to different genomic locations via an RNA intermediate. They are present in the genomes of virtually all eukaryotes and can be subdivided into two general structural classes. Long terminal repeat (LTR) retrotransposons resemble simple retroviruses but lack a functional envelope (Env) gene (2). Non-LTR retrotransposons lack LTRs and generally terminate in a polyadenylic acid [poly(A)] tail (20,23).L1s are the most abundant non-LTR retrotransposons in the human genome and comprise approximately 17% of nuclear DNA (42). The overwhelming majority of L1s are retrotransposition defective (RD-L1s) and cannot retrotranspose because they are 5Ј truncated, internally rearranged, or mutated (23); however, an estimated 30 to 60 human L1s remain retrotransposition competent (RC-L1s) (40). RC-L1s are 6.0 kb in length and contain a 5Ј untranslated region (UTR) harboring an internal promoter (43), two nonoverlapping open reading frames (open reading frame 1 [ORF1] and ORF2) (7, 41), and a 3Ј UTR ending in an unorthodox poly(A) tail (20,46). In addition, these elements are flanked by variable-length target site duplications, which are hallmarks of the retrotransposition process (20).Non-LTR retrotransposons encode endonuclease activities, which can generate either site-specific (4, 11, 47) or relatively non-site-specific nicks in chromosomal DNA (5, 10). The liberated 3Ј hydroxyl residue then acts as a primer for reverse transcription of the retrotransposon RNA by the retrotransposon-encoded reverse transcriptase (RT) by a mechanism termed target site-primed reverse transcription (TPRT) (28, 29). Thus, the processes of integration and reverse transcription are coupled for non-LTR retrotransposons.Biochemical studies revealed that ORF1 encodes a 40-kDa RNA binding protein that colocalizes with L1 RNA in cytoplasmic ribonucleoprotein particles (RNPs) (17, 18). ORF2 encodes a multifunctional protein containing endonuclease and RT activities (10, 34) and also has a carboxyl-terminal cysteine-rich domain (C) of unknown function (9). Using an assay to monitor L1 ret...
Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.
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