Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PAN^K217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging since changes can occur simultaneously at protease, their inhibitor and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics and peptidase predictions for studying proteolytic events in the saliva of seventy-nine patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (ROC-AUC>0.85). In addition, endo- and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from publicly repositories reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis was further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by SRM-MS, whilst minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of OSCC patients with lymph node metastasis and, at least in part, this is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis.

A black bear with a sweet tooth visited the front porch of a Connecticut home to raid a bowl of Halloween candy and steal a pumpkin.

Software implementation in high-performance computing is getting more fragmented as organizations opt for tools in their walled garden environments.  However, a new organization formed under the Linux Foundation could bring […]

The post Public and Private Sectors Team up to Solve HPC Software Problem  appeared first on HPCwire.

March 14, 2024 — Keysight Technologies, Inc. and Q-CTRL are partnering to integrate key infrastructure quantum software to accelerate quantum processor development, characterization, and key proof of principle scientific demonstrations. An interesting […]

The post Keysight and Q-CTRL Team Up to Accelerate Infrastructure Quantum Software appeared first on HPCwire.

An arms contractor is making "good progress" on the production of five B-21 Raider stealth bombers, U.S. Air Force Secretary Frank Kendall disclosed during a speech on Monday.

Customers now have access to Google’s homegrown hardware — its Axion CPU and latest Trillium TPU — in its Cloud service.  At the same time, Google gave customers a teaser […]

The post Role Reversal: Google Teases Nvidia’s Blackwell as It Softens TPU Rivalry appeared first on HPCwire.

HPC news headlines often highlight the latest hardware speeds and feeds. While advances on the hardware front are important, improving the ability to write software for advanced systems is equally […]

The post What’s New with Chapel? Nine Questions for the Development Team appeared first on HPCwire.

Nov. 6, 2024 — Harnessing the power artificial intelligence (AI) and the world’s fastest supercomputers, a research team led by the U.S. Department of Energy’s (DOE) Argonne National Laboratory has developed […]

The post Argonne Team Breaks New Ground in AI-driven Protein Design appeared first on HPCwire.

Nov. 2, 2023 — Researchers from the Argonne Leadership Computing Facility (ALCF), the University of St. Thomas, and the University of Illinois Chicago (UIC) won first place and “best workflow” […]

The post ALCF Team Wins 1st Place and Best Workflow at 2023 IEEE SciVis Contest appeared first on HPCwire.

A new HBO Max teaser offers a glimpse of the upcoming shows viewers can look forward to in the next year.

Paramount Pictures is teasing "Mission: Impossible -- The Final Reckoning," starring Tom Cruise.

"Alien: Earth," "Andor" Season 2, "The Bear" Season 4, "Daredevil: Born Again" and other Disney+ and Hulu series will premiere in 2025.

Hulu released the trailer for "Nutcrackers" Tuesday. The Ben Stiller holiday comedy premieres Nov. 29.

Here's a wild stat: nearly one-third of the world's food production goes to waste each year, a major contributor to the climate crisis. Farmer and UN climate champion Gonzalo Muñoz sheds light on the international negotiations aimed at turning the food system into a climate solution, rather than part of the problem — and shows the progress already underway.

Schools must make the critical decision whether to reinforce the learning that students have already done this year or introduce new content.

The U.S. Supreme Court’s recent ruling was a victory, but it doesn’t take away the hurt of losing my students in a cold February, writes Brett Bigham.

Teachers in North Carolina and Oregon plan to walk out of their classrooms in protest in May.

High school teacher Jenefer Pasqua is running for Wyoming's state legislature to fight against education funding cuts.

More schools are trying to shift to some in-person learning for students, but many teachers don’t believe it’s safe.

The pandemic has made it harder for teachers to access the moral rewards of their work, writes education professor Doris A. Santoro.

After a summer of protests over racial injustice, school districts are embracing anti-bias programs. The problem is: Few studies show they work.

As the bizarre becomes ordinary, don't forget what's at stake for America's teachers during the COVID-19 pandemic, writes Justin Minkel.

Schools and staff confront a welter of employment laws and regulations when it comes to on-site work under the pandemic.

Marc Tucker discusses the work of the Maryland Commission on Excellence and Innovation in Education, with support from NCEE, to update the state's school funding formula and to develop policies and practices to match the performance of the world's top-performing education systems.

Even when schools remain open, teachers across the country are speaking out for an investment in public education and protesting school-choice measures.

Thousands of teachers marched in Annapolis on Monday evening, pushing for higher pay and more resources for their students.

While most teachers have online access at home, internet service for many educators in rural areas is spotty, expensive, or nonexistent.

Too often, teachers are asked to use SEL practices without enough training and ongoing support, tanking the effectiveness.

Massachusetts' new performance assessment for teacher candidates helps boost readiness.

The statewide email to teachers is just the latest chapter in a rocky relationship between Kentucky officials and teachers.

At least four Kentucky school districts were forced to close last Thursday as hundreds of teachers called in sick to continue protesting what they believe to be anti-public education proposals in the state legislature.

Several school districts have been forced to close due to a large number of teachers calling out sick in protest of a pension-reform bill.