A Tale of Two Proteins: The Budding Yeast STUbL Subunit Slx5 Functionally Interacts with the SUMO Ligase Siz1

Eukaryotic cells utilize the dynamic addition and removal of post-translational modifications to modulate protein function. Two such modifiers are ubiquitin and SUMO (Small Ubiquitin-like MOdifier), which traditionally regulate their substrates in opposite ways. The discovery of SUMO-targeted ubiquitin ligases (STUbLs), E3 ligases that ubiquitylate sumoylated targets, offers an opportunity for cross-talk between the SUMO and ubiquitin pathways. STUbLs are crucial for the response to DNA damage and maintenance of genomic integrity, but currently only a few STUbL substrates are known. Recently, we observed a novel interaction between the yeast STUbL subunit Slx5 and the SUMO ligase Siz1 both in a yeast two-hybrid system and in vitro. The goals of this study were to develop protein extraction and purification protocols for the purpose of determining if Slx5 and Siz1 also interact in vivo. This study additionally seeks to determine if Siz1 is a target for ubiquitylation by Slx5 in vivo. Here we report our finding that Slx5 and Siz1Δ440 co-affinity purify in in vivo pulldown experiments, and that Siz1Δ440 is ubiquitylated in vivo in an Slx5-dependent manner. We also describe the intrinsic binding ability of the RING domain present in E3 ligases for metal affinity purification

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5

Pulmonary Nontuberculous Mycobacterial Infection. A Multisystem, Multigenic Disease

Rationale: The clinical features of patients infected with pulmonary nontuberculous mycobacteria (PNTM) are well described, but the genetic components of infection susceptibility are not

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

MPEG1/perforin-2 mutations in human pulmonary nontuberculous mycobacterial infections

Perforin-2 is a highly conserved pore-forming protein encoded by macrophage expressed gene 1 (MPEG1). A number of studies have shown that Perforin-2-deficient mice are unable to survive following a bacterial challenge that is nonlethal in WT mice. There is also recent evidence that Mpeg1+/- heterozygous mice display an intermediate killing ability compared with Mpeg1 WT and Mpeg1-/- mice. Despite these in vivo findings, to date, no perforin-2 deficiencies have been associated with human disease. Here, we report four patients with persistent nontuberculous mycobacterial infection who had heterozygous MPEG1 mutations. In vitro, neutrophils, macrophages, and B cells from these patients were unable to kill Mycobacterium avium as efficiently as normal controls. CRISPR mutagenesis validated the deleterious antibacterial activity of these mutations. These data suggest that perforin-2 haploinsufficiency may contribute to human susceptibility to infections with intracellular bacteria

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns. Keywords: CAR-T, dimerizer, cell therapy, costimulation switch, apoptosis, rapamycin, rimiducid, iMC, iRC9, safety switc

Pulmonary nontuberculous mycobacterial infection a multisystem, multigenic disease

Rationale: The clinical features of patients infected with pulmonary nontuberculous mycobacteria (PNTM) are well described, but the genetic components of infection susceptibility are not