Article Watch: September, 2023.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Article Watch: July, 2023.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Article Watch: July, 2022.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Article Watch: September, 2022.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Article Watch: December, 2022.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Article Watch: December 2017.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: September 2017.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail; cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: July 2017.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: April 2017.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: December 2016.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: September 2016.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

Article Watch: July 2016.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the association.

A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein ß activation and protects from inflammatory disease.

Toll-like receptors (TLRs) are expressed on innate immune cells and trigger inflammation upon detection of pathogens and host tissue injury. TLR-mediated proinflammatory-signaling pathways are counteracted by partially characterized anti-inflammatory mechanisms that prevent exaggerated inflammation and host tissue damage as manifested in inflammatory diseases. We biochemically identified a component of TLR-signaling pathways, A20-binding inhibitor of NF-κB (ABIN1), which recently has been linked by genome-wide association studies to the inflammatory diseases systemic lupus erythematosus and psoriasis. We generated ABIN1-deficient mice to study the function of ABIN1 in vivo and during TLR activation. Here we show that ABIN1-deficient mice develop a progressive, lupus-like inflammatory disease characterized by expansion of myeloid cells, leukocyte infiltrations in different parenchymatous organs, activated T and B lymphocytes, elevated serum Ig levels, and the appearance of autoreactive antibodies. Kidneys develop glomerulonephritis and proteinuria, reflecting tissue injury. Surprisingly, ABIN1-deficient macrophages exhibit normal regulation of major proinflammatory signaling pathways and mediators but show selective deregulation of the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) and its target genes, such as colony-stimulating factor 3 (Csf3), nitric oxide synthase, inducible (Nos2), and S100 calcium-binding protein A8 (S100a8). Their gene products, which are intimately linked to innate immune cell expansion (granulocyte colony-stimulating factor), cytotoxicity (inducible nitric oxide synthase), and host factor-derived inflammation (S100A8), may explain, at least in part, the inflammatory phenotype observed. Together, our data reveal ABIN1 as an essential anti-inflammatory component of TLR-signaling pathways that controls C/EBPß activity.

Bub1-mediated adaptation of the spindle checkpoint.

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore-microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest.

Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome.

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.

Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome.

The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.

Simultaneous measurement of intracellular triphosphate metabolites of zidovudine, lamivudine and abacavir (carbovir) in human peripheral blood mononuclear cells by combined anion exchange solid phase extraction and LC-MS/MS.

All nucleoside reverse transcriptase inhibitors (NRTI) must first be metabolized to their triphosphate forms in order to be active against HIV. Zidovudine (ZDV), abacavir (ABC) and lamivudine (3TC) have proven to be an efficacious combination. In order simultaneously to measure intracellular levels of the triphosphates (-TP) of ZDV, ABC (carbovir, CBV) and 3TC, either together or individually, we have developed a cartridge-LC-MS/MS method. The quantitation range was 2.5-250 pg/microl for 3TC-TP, 0.1-10.0 pg/microl for ZDV-TP and 0.05-5.00 pg/microl for CBV-TP. This corresponds to 0.1-11.0 pmol 3TC-TP per million cells, 4-375 fmol ZDV-TP per million cells and 2-200 fmol CBV-TP per million cells, extracted from 10 million cells. Patient samples demonstrated measured levels in the middle regions of our standard curves both at pre-dose and 4h post-dose times.

Proteomic studies of the intrinsically unstructured mammalian proteome.

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.