Duncan L. Smith

Latency-Associated Degradation of the MRP1 Drug Transporter During Latent Human Cytomegalovirus Infection

Hide-and-Seek Virus Human cytomegalovirus (HCMV) establishes latent infection in human progenitor dendritic cells, causing significant morbidity and mortality on reactivation, which may occur in transplantation patients who are immunosuppressed. Neither detection nor selective removal of rare latent HCMV-infected cells has been possible. Weekes et al. (p. 199) have found that the multidrug-resistant ABC transporter, multidrug resistance–associated protein-1 (MRP1) is down-regulated during latent HCMV infection. Consequently, cytotoxic MRP1-specific substrates are not exported from HCMV-infected cells and accumulate—leading to cell death, which could potentially provide a mechanism for eliminating infected cells prior to transplantation. A virally encoded protein eliminates a cell surface drug transporter, promoting latent human cytomegalovirus infection. The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture–based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance–associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14+ and CD34+ progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.

A PP1-PP2A phosphatase relay controls mitotic progression

The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1–cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.

Quantitative Proteomics Analysis Demonstrates Post-transcriptional Regulation of Embryonic Stem Cell Differentiation to Hematopoiesis

Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of BryGFP/+ ES cell to hemangioblasts can be followed by the expression of the BryGFP/+ and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating BryGFP ES cells were obtained by flow cytometric sorting, GFP−Flk1− (epiblast), GFP+Flk1− (mesoderm), and GFP+Flk1+ (hemangioblast). Microarray analyses and relative quantification two-dimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level.

Mitochondrial mass, a new metabolic biomarker for stem-like cancer cells: Understanding WNT/FGF-driven anabolic signaling.

Here, we developed an isogenic cell model of “stemness” to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/β-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine. Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population. The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

Intramembrane proteolytic cleavage by signal peptide peptidase is required for the turnover of some ER-resident, tail-anchored membrane proteins.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10(+)/CD19(+) fraction of bone marrow aspirates obtained from relapsed compared with normal controls and those with primary disease. CD10(+)/CD19(+) fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS.

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel‐separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid‐phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid‐labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two‐dimensional (2‐D) PAGE separation and MALDI‐MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2‐D PAGE. Manual and automated procedures for spot cutting and in‐gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI‐MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.

HUWE1 Ubiquitylates and Degrades the RAC Activator TIAM1 Promoting Cell-Cell Adhesion Disassembly, Migration, and Invasion

Summary The E3 ubiquitin ligase HUWE1, deregulated in carcinoma, has been implicated in tumor formation. Here, we uncover a role for HUWE1 in cell migration and invasion through degrading the RAC activator TIAM1, implying an additional function in malignant progression. In MDCKII cells in response to HGF, HUWE1 catalyzes TIAM1 ubiquitylation and degradation predominantly at cell-cell adhesions, facilitating junction disassembly, migration, and invasion. Depleting HUWE1 or mutating the TIAM1 ubiquitylation site prevents TIAM1 degradation, antagonizing scattering, and invasion. Moreover, simultaneous depletion of TIAM1 restores migration and invasion in HUWE1-depleted cells. Significantly, we show that HUWE1 stimulates human lung cancer cell invasion through regulating TIAM1 stability. Finally, we demonstrate that HUWE1 and TIAM1 protein levels are inversely correlated in human lung carcinomas. Thus, we elucidate a critical role for HUWE1 in regulating epithelial cell-cell adhesion and provide additional evidence that ubiquitylation contributes to spatiotemporal control of RAC.

Augmented annotation of the Schizosaccharomyces pombe genome reveals additional genes required for growth and viability.

Genome annotation is a synthesis of computational prediction and experimental evidence. Small genes are notoriously difficult to detect because the patterns used to identify them are often indistinguishable from chance occurrences, leading to an arbitrary cutoff threshold for the length of a protein-coding gene identified solely by in silico analysis. We report a systematic reappraisal of the Schizosaccharomyces pombe genome that ignores thresholds. A complete six-frame translation was compared to a proteome data set, the Pfam domain database, and the genomes of six other fungi. Thirty-nine novel loci were identified. RT-PCR and RNA-Seq confirmed transcription at 38 loci; 33 novel gene structures were delineated by 5′ and 3′ RACE. Expression levels of 14 transcripts fluctuated during meiosis. Translational evidence for 10 genes, evolutionary conservation data supporting 35 predictions, and distinct phenotypes upon ORF deletion (one essential, four slow-growth, two delayed-division phenotypes) suggest that all 39 predictions encode functional proteins. The popularity of S. pombe as a model organism suggests that this augmented annotation will be of interest in diverse areas of molecular and cellular biology, while the generality of the approach suggests widespread applicability to other genomes.