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.

When the drug Taxol® was approved by the United States Food and Drug Administration in 1993, it was a game changer for cancer patients. The compound, which arrests cell division by preventing the disassembly of tubulin microfibers, has been used over the past three decades to treat millions of cases of breast, lung, and ovarian cancer as well as Kaposi's sarcoma. In 1990, Bristol Myers Squibb applied to trademark the name Taxol, which was approved in 1992, changing the drug's generic name to paclitaxel.At the time that Taxol was entering clinical trials in the late 1970s, it also proved to be a valuable tool for cytoskeletal research. Tubulin had been discovered in the late 1960s, but it was still unclear how the soluble protein dimer polymerized (Fig. 1) to form the long, complex structures of the cytoskeleton.jbc;295/41/13994/F1F1F1Figure 1.Strands of tubulin, a protein in the cell's skeleton, photographed using a high-resolution microscopy technique. Image made by Pakorn Kanchanawong (National University of Singapore) and Clare Waterman (NHLBI, National Institutes of Health).“Back then, people were just discovering the most basic functions of tubulin and how it polymerized, and then they found a drug that affected this,” said Velia Fowler, a cell biologist at the University of Delaware and former Associate Editor at the Journal of Biological Chemistry.The drug and its cytoskeletal activity intersected in the 1981 JBC paper “Taxol-induced polymerization of purified tubulin” (1), the subject of this JBC Classic. In the single-author paper, Nirbhay Kumar, then a postdoctoral fellow at the National...

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
Research Articles

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

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

Melissa Gómez
Dec 1, 2020; 61:1658-1674
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

Chiara Pavanello
Dec 1, 2020; 61:1784-1788
Patient-Oriented and Epidemiological Research

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

Daphne E.C. Boer
Dec 23, 2020; 0:jlr.RA120001043v1-jlr.RA120001043
Research Articles

Douglas A. Andres
Dec 1, 2020; 61:1537-1537
Images in Lipid Research

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

Ryunosuke Ohkawa
Dec 1, 2020; 61:1577-1588
Research Articles

Genta Kakiyama
Dec 1, 2020; 61:1629-1644
Research Articles

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

Michael E. Abrams
Dec 1, 2020; 61:1538-1538
Images in Lipid Research

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

Thibaut Bourgeois
Dec 11, 2020; 0:jlr.RA120000737v1-jlr.RA120000737
Research Articles

Julia V Busik
Dec 23, 2020; 0:jlr.TR120000981v1-jlr.TR120000981
Thematic Reviews

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

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

Abudukadier Abulizi
Dec 1, 2020; 61:1565-1576
Research Articles

Ying Zou
Dec 1, 2020; 61:1589-1604
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

Jenny E. Kanter
Dec 8, 2020; 0:jlr.ILR120001217v1-jlr.ILR120001217
Images in Lipid Research

Yueming Hu
Dec 11, 2020; 0:jlr.RA120000919v1-jlr.RA120000919
Research Articles

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

Stacey N Keenan
Dec 17, 2020; 0:jlr.RA120001126v1-jlr.RA120001126
Research Articles

Akemi Kakino
Nov 3, 2020; 0:jlr.RA120000767v1-jlr.RA120000767
Research Articles

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

Amanda D.V. MacCannell
Nov 18, 2020; 0:jlr.ILR120001204v1-jlr.ILR120001204
Images in Lipid Research

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

For data that is being loaded from a SAS Stored Process Server, an insertion process might fail to a MySQL database with a warning, as well as an error message that says "During insert: Incorrect datetime value…"

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, you might notice that you cannot clear warnings for decision campaign nodes by selecting either the Clear Warnings  option or the Clear All Warnin

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

You encounter this issue when the table metadata matches the data source. In this scenario, no metadata update is required.

In SAS Business Rules Manager 3.3, the initial loading of a rule set and a rule flow takes significantly longer than it does in release 3.2. When this problem happens, long time gaps are evident in the local