Faculty Bibliography

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

Objectives: Ovarian cancer is the most lethal gynecologic malignancy. Following the initial response to platinum drugs, most patients experience recurrence of resistant disease, necessitating development of durable new treatments. Resistance to treatment is centered on cancer stem (initiating) cells (CSCs) and the AKT pathway. AKT regulates mTOR complex (mTORC1/2), downstream mRNA translation, cell proliferation, survival, and angiogenesis in tumorigenesis. Rapalogs are clinically approved inhibitors of mTORC1 but have no effect on mTORC2. We investigate a clinical experimental dual mTORC1/2 inhibitor on platinum-resistant ovarian CSCs.Methods: Platinum-resistant ovarian OVCAR3 cells were treated with vehicle, carboplatin, or INK128/MLN128, (mTORC1/2 inhibitor). CSCs were identified with flow cytometry or isolated using fluorescence-activated cell sorting using CD133 +/CD44 +. The CSC population was quantified using non-adherent spheroid growth assays. Cell viability was determined by colony formation assays with spheroids for carboplatin re-sensitization by mTORC1 or mTORC1/2 inhibition. Doxycycline-inducible shRNA silencing cell lines were generated from OVCAR3 cells to inhibit Raptor (mTORC1) or Rictor proteins (mTORC2).Results: CSCs comprise 4.9% of untreated OVCAR3 cells, confirmed by stem cell markers Oct4 and Sox2, and AKT/mTORC1 activation by phosphorylated-4E-BP1. Surprisingly, INK128 treatment increased the platinum-resistant CSC population by 2-fold (Fig. 1, P <.005), whereas carboplatin alone had no effect. INK128-treated cells formed fewer colonies compared with vehicle controls (mean colony number 36.3 + 23.8 vs 83.3 + 32.1, P <.001) and smaller colonies (mean cell number 5,260 + 835.8 vs 6,907 + 702.4, P <.0002). NS- and Raptor-silenced cells were unchanged in all assays, whereas Rictor silencing reduced CSCs by half (Fig. 2, P <.002) and formed fewer colonies by approximately 5-fold (P <.03).Conclusions: The PI3K/AKT/mTOR pathway promotes platinum resistance in ovarian cancer cells, and supports the CSC population, but can be overcome by pharmacologic inhibition of mTORC1/2. Drugging mTORC1/2 does not selectively inhibit CD133 +/CD44 + ovarian CSCs, but appears to target non-CSCs and "stem-like" cells based on functional data. Targeting mTORC1/2 sensitizes the resistant CSC population and should be clinically explored. (figure present)

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA/dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions.

The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs

Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the alpha-subunit of translation initiation factor 2 (eIF2alpha) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP(-/-) cells exposed to perturbations that impair ER function. CHOP(-/-) and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2alpha dephosphorylation, like CHOP(-/-) mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1alpha, which encodes an ER oxidase. Consequently, the ER of stressed CHOP(-/-) cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium. Recently, apoLp-III in Galleria mellonella larvae was shown to play an unexpected role in insect immune activation. We identified the cDNA sequence of Hyphantria cunea apoLp-III by oligonucleotide-primed amplification, and 5'- and 3'-RACE PCR. Since H. cunea has an unusually low level of apoLp-III in the hemolymph, a recombinant apoLp-III was overexpressed using a baculovirus expression system to investigate its biological activity. Recombinant apoLp-III and/or Escherichia coli were injected into the hemocoel of last instar larvae, and the expression of antimicrobial peptide from fat body was determined by Northern blot. Injection of apoLp-III as well as E. coli induced slight up-regulation of its transcription rate in fat body, whereas the expression of antimicrobial peptide was dramatically induced by the injection of apoLp-III and E. coli. H. cunea hemocytes had apoLp-III in the granules and expressed its transcript, albeit at a much lower level than in the fat body. Upon bacterial injection, a subpopulation of hemocytes showed degranulation and degradation. Local discharge of apoLp-III from hemocytes caused by the injection of E. coli might be related to the immune response through an unknown mechanism

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor

Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (alpha subunit and beta subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer (alpha1beta1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 degrees C. PCR amplification of these two subunits of PCD4,5 revealed that the alpha subunit and beta subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation