RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5' end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Alice Santonastaso
Dec 1, 2020; 61:1687-1696
Research Articles

Stephanie E. Hood
Dec 1, 2020; 61:1720-1732
Research Articles

Jiayan Guo
Dec 1, 2020; 61:1764-1775
Research Articles

Martin Prescher
Dec 1, 2020; 61:1605-1616
Research Articles

Frank C. Nichols
Dec 1, 2020; 61:1645-1657
Research Articles

Hideaki Kuge
Dec 1, 2020; 61:1747-1763
Research Articles

Elisa Vidal
Dec 1, 2020; 61:1733-1746
Research Articles

Marco De Giorgi
Dec 1, 2020; 61:1675-1686
Research Articles

Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
Research Articles

Prabuddha Dey
Dec 1, 2020; 61:1556-1564
Reviews

Nicholas D. LeBlond
Dec 1, 2020; 61:1697-1706
Research Articles

Auguste Dargent
Dec 1, 2020; 61:1776-1783
Research Articles

Astrid M. Moerman
Dec 23, 2020; 0:jlr.RA120000974v1-jlr.RA120000974
Research Articles

Natalie Bruiners
Dec 1, 2020; 61:1617-1628
Research Articles

Junhwan Kim
Dec 1, 2020; 61:1707-1719
Research Articles

Aloïs Dusuel
Dec 9, 2020; 0:jlr.RA120000704v1-jlr.RA120000704
Research Articles

Anja Jaeschke
Dec 9, 2020; 0:jlr.RA120001141v1-jlr.RA120001141
Research Articles

Oktawia Nilsson
Nov 17, 2020; 0:jlr.RA120000920v1-jlr.RA120000920
Research Articles

Tommaso Laurenzi
Dec 2, 2020; 0:jlr.RA120000843v1-jlr.RA120000843
Research Articles

Carlota Oleaga
Nov 17, 2020; 0:jlr.RA120000964v1-jlr.RA120000964
Research Articles

Babunageswararao Kanuri
Nov 17, 2020; 0:jlr.RA120001101v1-jlr.RA120001101
Research Articles

Gerhard Liebisch
Dec 1, 2020; 61:1539-1555
Special Report

In SAS Customer Intelligence Studio, you might notice that the user interface becomes unresponsive, as shown below: imgalt="SAS Customer Intelligence Studio UI becomes unresponsive" src="{fusion_66537

In SAS Customer Intelligence Studio, you can choose to create a new calculated variable in the Edit Value dialog box when you populate a treatment custom detail. Following creation of the new calculated

In SAS Customer Intelligence Studio, the following error is displayed when you update a new Link  node in a diagram:   imgalt="Link: MAIQService:executeFastPath:" src="{fusion_665

If you have configured more than one SAS Application Server, then SAS Visual Data Builder might unexpectedly use the wrong application server when you preview or schedule queries. This problem occurs even though you h

You might see the following error messages: "ERROR: Connection failed. Server returned: SAS Logon Manager authentication failed: Access denied." and "ERROR: Unable to connect to Cloud Analytic Services host-name on port 5570. Veri

If the response variable is in the CLASS statement variable list before the class variables that also appear in the MODEL statement, and an OM-data-set is used, least squares means results for several of the statistical procedures are incorrect.

You might receive a Segmentation Violation when opening a database table in SAS. The SAS Log contains the error and traceback:

In SAS Life Science Analytics Framework 4.6, group membership removal fails with an exception if a user is set as assignee, a candidate, or a notification recipient in a user task for a Process Flow . The Process

An error might occur when you use the SAS 9 BULKLOAD= and BULKEXTRACT= options load data to or extract data from HP Neoview on the HP Itanium platform. The problem occurs because Hewlett-Packard changed the name of one of

In SAS Merchandise Financial Planning, custom time frame-based data versions do not aggregate correctly when referenced in worksheets with standard hierarchy levels. The data does not aggregate correctly from l

In SAS Retail Space Management, it should be possible to click on any location object, then Show Properties, and change the location fill color. This can be done on the gray-filled objects. However, w

If the OBSMARGINS= or OM= option is specified in an LSMEANS, LSMESTIMATE, or SLICE statement and a user-defined format is applied to any of the effect variables in the OM-data-set , PROC PLM incorrectly stops proce

When viewing a SAS Visual Analytics report, you might intermittently see an error that includes content similar to the following:

In SAS Customer Intelligence Studio,  when you add  Reply- node variable values in the Define Value dialog box, you might notice that two identically labeled data-grid variables are

In SAS Infrastructure for Risk Management, the message "ERROR: Could not move and link one or more files to..." occurs while running a job-flow instance if an orphaned folder exists in the persistent area.

SAS Web Report Studio enables you to link reports based on a group break value. However, when you click the link, it might fail with an HTTP 400 error. The exact message you see depends on which browser you are u

When you log on to the SAS Risk Governance Framework and choose a solution, the web application might fail to load the solution content. When the problem occurs, you continue to see "Loading..." on the screen, an

Certain tables that are created in SAS Scalable Performance Data (SPD) Server might not be displayed correctly by SAS Federation Server Manager. Tables that have Latin5 characters in column names encounter this

This manuscript has been withdrawn by the Author.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Photoreceptors have high energy-demands and a high density of mitochondria that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) of fuel substrates. Although glucose is the major fuel for central nervous system (CNS) brain neurons, in photoreceptors (also CNS), most glucose is not metabolized through OXPHOS but is instead metabolized into lactate by aerobic glycolysis. The major fuel sources for photoreceptor mitochondria remained unclear for almost six decades. Similar to other tissues (like heart and skeletal muscle) with high metabolic rates, photoreceptors were recently found to metabolize fatty acids (palmitate) through OXPHOS. Disruption of lipid entry into photoreceptors leads to extracellular lipid accumulation, suppressed glucose transporter expression, and a duel lipid/glucose fuel shortage. Modulation of lipid metabolism helps restore photoreceptor function. However, further elucidation of the types of lipids used as retinal energy sources, the metabolic interaction with other fuel pathways, as well as the crosstalk among retinal cells to provide energy to photoreceptors is not yet known. In this review, we will focus on the current understanding of photoreceptor energy demand and sources, and potential future investigations of photoreceptor metabolism.