Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis.

Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. Previously, we showed that cell size control involves both cell size checkpoints, which delay cell cycle progression in small cells, and size-dependent regulation of mass accumulation rates (Ginzberg et al., 2018).

Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein.

Assembly and function of branched ubiquitin chains.

Post-translational modification with ubiquitin is required for cell division, differentiation, and survival in all eukaryotes. As part of an intricate signaling code, ubiquitin is attached to its targets as single molecules or polymeric chains, with the distinct modifications encoding a wide range of outcomes. After early work focused on homotypic ubiquitin chains, such as the K48-linked polymers that drive proteasomal degradation, recent studies noted abundant conjugates that contained ubiquitin molecules modified on two or more sites.

Ubiquitin-dependent regulation of transcription in development and disease.

Transcription is an elaborate process that is required to establish and maintain the identity of the more than two hundred cell types of a metazoan organism. Strict regulation of gene expression is therefore vital for tissue formation and homeostasis. An accumulating body of work found that ubiquitylation of histones, transcription factors, or RNA polymerase II is crucial for ensuring that transcription occurs at the right time and place during development.

Gene expression and cell identity controlled by anaphase-promoting complex.

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity.

Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence.

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d (labeled-Des) as an internal standard in different biological fluids, such as urine and serum.

EMI1 switches from being a substrate to an inhibitor of APC/C to start the cell cycle.

Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/C, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/C, which suggests that APC/C inactivation is the point of no return for cell-cycle entry .

Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge.

Prb1 Protease Activity Is Required for Its Recognition by the F-Box Protein Saf1.

The SCF ubiquitin ligase associates with substrates through its F-box protein adaptor. Substrates are typically recognized through a defined phosphodegron. Here, we characterize the interaction of the F-box protein Saf1 with Prb1, one of its vacuolar protease substrates.

Ndd1 turnover by SCF(Grr1) is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage.

Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.

Rationale: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times.

Methods: A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model.

Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins.

We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin-associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using ligase traps of eight different F box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously unreported, substrates.

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells.

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment.

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins.

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported.

Chemical genetics reveals a kinase-independent role for protein kinase R in pyroptosis.

Formation of the inflammasome, a scaffolding complex that activates caspase-1, is important in numerous diseases. Pyroptotic cell death induced by anthrax lethal toxin (LT) is a model for inflammasome-mediated caspase-1 activation. We discovered 7-desacetoxy-6,7-dehydrogedunin (7DG) in a phenotypic screen as a small molecule that protects macrophages from LT-induced death.

Identification of novel host-targeted compounds that protect from anthrax lethal toxin-induced cell death.

Studying how pathogens subvert the host to cause disease has contributed to the understanding of fundamental cell biology. Bacillus anthracis, the causative agent of anthrax, produces the virulence factor lethal toxin to disarm host immunity and cause pathology. We conducted a phenotypic small molecule screen to identify inhibitors of lethal toxin-induced macrophage cell death and used an ordered series of secondary assays to characterize the hits and determine their effects on cellular function.

Design, synthesis, and evaluation of 2-(arylsulfonyl)oxiranes as cell-permeable covalent inhibitors of protein tyrosine phosphatases.

A structure-based design approach has been applied to develop 2-(arylsulfonyl)oxiranes as potential covalent inhibitors of protein tyrosine phosphatases. A detailed kinetic analysis of inactivation by these covalent inhibitors reveals that this class of compounds inhibits a panel of protein tyrosine phosphatases in a time- and dose-dependent manner, consistent with the covalent modification of the enzyme active site. An inactivation experiment in the presence of sodium arsenate, a known competitive inhibitor of protein tyrosine phosphatase, indicated that these inhibitors were active site bound.

Stat3 mediates expression of autotaxin in breast cancer.

We determined that signal transducer and activator of transcription 3 (Stat3) is tyrosine phosphorylated in 37% of primary breast tumors and 63% of paired metastatic axillary lymph nodes. Examination of the distribution of tyrosine phosphorylated (pStat3) in primary tumors revealed heterogenous expression within the tumor with the highest levels found in cells on the edge of tumors with relatively lower levels in the central portion of tumors. In order to determine Stat3 target genes that may be involved in migration and metastasis, we identified those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels.

Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas.

Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis.

Coulomb effects in binding of heme in gas-phase ions of myoglobin.

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry.

Comparison of automated and manual MRI volumetry of hippocampus in normal aging and dementia.

Purpose: To determine whether automatic and manual measurements of hippocampal volume differences on MRI between normal aging, cognitive impairment (CI), and Alzheimer's disease (AD) yield similar results.

Materials And Methods: Reliability was determined for an automatic and a manual method on nine volunteers (22-83 years old) who underwent MRI twice in 1 day. Hippocampal volumes of 20 cognitively normal subjects (mean age 74.