A genetic strategy involving a glycosyltransferase promoter and a lipid translocating enzyme to eliminate cancer cells

The most common therapeutic strategy for the treatment of cancer uses antimetabolites, which block uncontrolled division of cancer cells and kill them. However, such antimetabolites also kill normal cells, thus yielding detrimental side effects. This emphasizes the need for an alternative therapy, which would have little or no side effects. Our approach involves designing genetic means to alter surface lipid determinants that induce phagocytosis of cancer cells. The specific target of this strategy has been the enzyme activity termed aminophospholipid translocase (APLT) or flippase that causes translocation of phosphatidylserine (PS) from the outer to the inner leaflet of the plasma membrane in viable cells. Efforts to identify the enigmatic, plasma membrane APLT of mammalian cells have led investigators to some P-type ATPases, which have often proven to be the APLT of internal membranes rather than the plasma membrane. By measuring kinetic parameters for the plasma membrane APLT activity, we have shown that the P-type ATPase Atp8a1 is the plasma membrane APLT of the tumorigenic N18 cells, but not the non-tumorigenic HN2 (hippocampal neuron × N18) cells. Targeted knockdown of this enzyme causes PS externalization in the N18 cells, which would trigger phagocytic removal of these cells. But how would we specifically express the mutants or antisense Atp8a1 in the cancer cells? This has brought us to a glycosyltransferase, GnT-V, which is highly expressed in the transformed cells. By using the GnT-V promoter to drive a luciferase reporter gene we have demonstrated a dramatic increase in luciferase expression selectively in tumor cells. The described strategy could be tested for the removal of cancer cells without the use of antimetabolites that often kill normal cells.     

Molecular Mechanisms of Rhodopsin Retinitis Pigmentosa and the Efficacy of Pharmacological Rescue

Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.     

The Physical and Genetic Organization of A Viral Genom

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Phosphatidylethanolamine as a prion cofactor

Mammalian prions with significant levels of specific infectivity can be formed in vitro from mixtures of prion protein (PrP) and cofactor molecules, but not from PrP alone. We recently isolated and identified the essential membrane phospholipid phosphatidylethanolamine (PE) as an endogenous cofactor for prion propagation in vitro.¹ Deleault NR, Piro JR, Walsh DJ, Wang F, Ma J, Geoghegan JC, et al. Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids. Proc Natl Acad Sci U S A 2012; 109:8546 - 51; http://dx.doi.org/10.1073/pnas.1204498109; PMID: 22586108 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] In this article, we discuss the potential role of PE and other essential cofactor molecules as a molecular link between the processes of prion formation and prion-induced neurodegeneration.     

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains

Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy.     

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.     

Phospholipid Transport via Mitochondria

In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes (OM and IM) with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. As cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial OM and IM. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.      

Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.     

Novel Aspects of the Acid Response Network of E. coli K-12 Are Revealed by a Study of Transcriptional Dynamics

Understanding gene regulation and its adaptive significance requires not only a detailed knowledge of individual molecular interactions that give rise to changes in gene expression but also an overview of complete genetic networks and the ways in which components within them interact. Increasingly, such studies are being done using luminescent or fluorescent reporter proteins that enable monitoring of gene expression dynamics in real time, particularly during changes in expression. We show here that such an approach is valid for dissecting the responses of the AR2 or GAD network of Escherichia coli K-12 to changes in pH, which is one of the most complex networks known in E. coli. In addition to confirming several regulatory interactions that have been revealed by previous studies, this approach has identified new components in this system that lead to complex dynamics of gene expression following a drop in pH, including an auto-regulatory loop involving the YdeO activator protein and novel roles for the PhoP protein.