The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes.

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino- or carboxyl-termini are eliminated by the N- or C-degron pathways, respectively. We aimed to elucidate the function of C-degron pathways and to unveil how normal proteomes are exempt from C-degron pathway-mediated destruction. Our data reveal that C-degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C-degron and N-degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C-degron pathways, but not of defective proteins, suggesting proteolysis-based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.

Differential Innate Immune Signaling in Macrophages by Wild-Type Vaccinia Mature Virus and a Mutant Virus with a Deletion of the A26 Protein.

The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of the A26 protein from the WR strain mature virus generates a mutant, WRΔA26, that enters HeLa cells through plasma membrane fusion. Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type strain WR and the WRΔA26 mutant and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies, in which both HeLa cells infected with WR and HeLa cells infected with WRΔA26 expressed abundant viral late proteins, we found that WR expressed much less viral late protein than WRΔA26 in BMDM. Microarray analysis of the cellular transcripts in BMDM induced by virus infection revealed that WR preferentially activated type 1 interferon receptor (IFNAR)-dependent signaling but WRΔA26 did not. We consistently detected a higher level of soluble beta interferon secretion and phosphorylation of the STAT1 protein in BMDM infected with WR than in BMDM infected with WRΔA26. When IFNAR-knockout BMDM were infected with WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WR and, in turn, restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRΔA26 infection than to that from WR infection, whereas IFNAR-knockout mice were equally susceptible to WR and WRΔA26 infection, demonstrating that the ability of WRΔA26 to evade IFNAR signaling has an important influence on viral pathogenesis in vivoIMPORTANCE The vaccinia virus A26 protein was previously shown to mediate virus attachment and to regulate viral endocytosis. Here, we show that infection with strain WR induces a robust innate immune response that activates type 1 interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas infection with the WRΔA26 mutant does not. We further demonstrated that the differential activation of IFNAR-dependent cellular signaling between WR and WRΔA26 not only is important for differential host restriction in BMDM but also is important for viral virulence in vivo Our study reveals a new property of WRΔA26, which is in regulating host antiviral innate immunity in vitro and in vivo.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Sodium Fluoride Exposure Leads to ATP Depletion and Altered RNA Decay in Escherichia coli under Anaerobic Conditions.

Although fluoride-containing compounds are widely used to inhibit bacterial growth, the reprogramming of gene expression underlying cellular responses to fluoride, especially under anaerobic conditions, is still poorly understood. Here, we compare the genome-wide transcriptomic profiles of E. coli grown in the absence (control) or presence (20 and 70 mM) of sodium fluoride (NaF) under anaerobic conditions and assess the impact of fluoride-dependent ATP depletion on RNA turnover. Tiling array analysis revealed transcripts displaying altered abundance in response to NaF treatments. Quantile-based K-means clustering uncovered a subset of genes that were highly upregulated and then downregulated in response to increased and subsequently decreased fluoride concentrations, many of which (~40%) contained repetitive extragenic palindromic (REP) sequences. Northern blot analysis of some of these highly upregulated REP-containing transcripts (i.e., osmC, proP, efeO and yghA) confirmed their considerably enhanced abundance in response to NaF treatment. An mRNA stability analysis of osmC and yghA transcripts demonstrated that fluoride treatment slows down RNA degradation, thereby enhancing RNA stability and steady-state mRNA levels. Moreover, we demonstrate that turnover of these transcripts depends on RNase E activity and RNA degradosome. Thus, we show that NaF exerts significant effects at the whole-transcriptome level under hypoxic growth (i.e., mimicking the host environment), and fluoride can impact gene expression posttranscriptionally by slowing down ATP-dependent degradation of structured RNAs. IMPORTANCE Gram-negative Escherichia coli is a rod-shaped facultative anaerobic bacterium commonly found in microaerobic/anaerobic environments, including the dental plaques of warm-blooded organisms. These latter can be treated efficiently with fluoride-rich compounds that act as anticaries agents to prevent tooth decay. Although fluoride inhibits microbial growth by affecting metabolic pathways, the molecular mechanisms underlying its activity under anaerobic conditions remain poorly defined. Here, using genome-wide transcriptomics, we explore the impact of fluoride treatments on E. coli gene expression under anaerobic conditions. We reveal key gene clusters associated with cellular responses to fluoride and define its ATP-dependent stabilizing effects on transcripts containing repetitive extragenic palindromic sequences. We demonstrate the mechanisms controlling the RNA stability of these REP-containing mRNAs. Thus, fluoride can affect gene expression posttranscriptionally by stabilizing structured RNAs.

FAM21 is critical for TLR2/CLEC4E-mediated dendritic cell function against Candida albicans.

FAM21 (family with sequence similarity 21) is a component of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) protein complex that mediates actin polymerization at endosomal membranes to facilitate sorting of cargo-containing vesicles out of endosomes. To study the function of FAM21 in vivo, we generated conditional knockout (cKO) mice in the C57BL/6 background in which FAM21 was specifically knocked out of CD11c-positive dendritic cells. BMDCs from those mice displayed enlarged early endosomes, and altered cell migration and morphology relative to WT cells. FAM21-cKO cells were less competent in phagocytosis and protein antigen presentation in vitro, though peptide antigen presentation was not affected. More importantly, we identified the TLR2/CLEC4E signaling pathway as being down-regulated in FAM21-cKO BMDCs when challenged with its specific ligand Candida albicans Moreover, FAM21-cKO mice were more susceptible to C. albicans infection than WT mice. Reconstitution of WT BMDCs in FAM21-cKO mice rescued them from lethal C. albicans infection. Thus, our study highlights the importance of FAM21 in a host immune response against a significant pathogen.

Identification of UAP1L1 as a critical factor for protein O-GlcNAcylation and cell proliferation in human hepatoma cells.

Aged hepatocyte-specific-Mcl-1 knockout (MKO-hep) mice are prone to develop liver tumors mimicking human hepatocellular carcinoma (HCC). Here we reported that a protein named UDP-N-acetylglucosamine pyrophosphorylase-1-like-1 (Uap1l1) is upregulated in the liver of young MKO-hep mice without any macroscopically detectable tumor nodules and is prominently expressed in the hepatic tumors developed in the aged MKO-hep mice. Intriguingly, human UAP1L1 is also significantly upregulated in a distinct subset of HCC tissues and patients with upregulated expression of UAP1L1 appeared to have poor prognosis. Overexpression of UAP1L1 significantly promoted, whereas UAP1L1 knockdown markedly reduced the proliferation of human hepatoma cells both in vitro and in vivo. UAP1L1 shows ~59% sequence identity to UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1), which is directly involved in the synthesis of the sugar donor (UDP-GlcNac) for N-acetylglucosamine modification (O-GlcNAcylation) of proteins. However, unlike UAP1, UAP1L1 harbors very limited UDP-GlcNAc synthesis activity. Moreover, although both UAP1 and UAP1L1 are required for O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation, they appear to function distinctly from each other. UAP1L1 directly interacts with OGT, but does not seem to be an OGT substrate. In addition, UAP1L1 alone is not sufficient to activate OGT activity in vitro, suggesting that UAP1L1 may function together with other proteins to modulate OGT activity in vivo. Lastly, UAP1L1 knockdown attenuated c-MYC O-GlcNAcylation and protein stability, and overexpression of c-MYC significantly rescued the proliferation defect of UAP1L1 knockdown HepG2 cells, suggesting that c-MYC is one downstream target of UAP1L1 that contributes to UAP1L1-mediated cell proliferation, at least in HepG2 cells.