Mechanisms and function of substrate recruitment by F-box proteins.

S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes use a family of F-box proteins as substrate adaptors to mediate the degradation of a large number of regulatory proteins involved in diverse processes. The dysregulation of SCF complexes and their substrates contributes to multiple pathologies. In the 14 years since the identification and annotation of the F-box protein family, the continued identification and characterization of novel substrates has greatly expanded our knowledge of the regulation of substrate targeting and the roles of F-box proteins in biological processes. Here, we focus on the evolution of our understanding of substrate recruitment by F-box proteins, the dysregulation of substrate recruitment in disease and potential avenues for F-box protein-directed disease therapies.

Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2.

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

mTOR generates an auto-amplification loop by triggering the ßTrCP- and CK1α-dependent degradation of DEPTOR.

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein ßTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the ßTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the ßTrCP-dependent degradation of DEPTOR via ßTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind ßTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and ßTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways.

SCF ubiquitin ligases in the maintenance of genome stability.

In response to genotoxic stress, eukaryotic cells activate the DNA damage response (DDR), a series of pathways that coordinate cell cycle arrest and DNA repair to prevent deleterious mutations. In addition, cells possess checkpoint mechanisms that prevent aneuploidy by regulating the number of centrosomes and spindle assembly. Among these mechanisms, ubiquitin-mediated degradation of key proteins has an important role in the regulation of the DDR, centrosome duplication and chromosome segregation. This review discusses the functions of a group of ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) family, in the maintenance of genome stability. Given that general proteasome inhibitors are currently used as anticancer agents, a better understanding of the ubiquitylation of specific targets by specific ubiquitin ligases may result in improved cancer therapeutics.

Systematic Literature Review of the Association of Fever and Elevated Temperature with Outcomes in Critically Ill Adult Patients.

Although most commonly associated with infection, elevated temperature and fever also occur in a variety of critically ill populations. Prior studies have suggested that fever and elevated temperature may be detrimental to critically ill patients and can lead to poor outcomes, but the evidence surrounding the association of fever with outcomes is rapidly evolving. To broadly assess potential associations of elevated temperature and fever with outcomes in critically ill adult patients, we performed a systematic literature review focusing on traumatic brain injury, stroke (ischemic and hemorrhagic), cardiac arrest, sepsis, and general intensive care unit (ICU) patients. Searches were conducted in Embase® and PubMed® from 2016 to 2021, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, including dual-screening of abstracts, full texts, and extracted data. In total, 60 studies assessing traumatic brain injury and stroke (24), cardiac arrest (8), sepsis (22), and general ICU (6) patients were included. Mortality, functional, or neurological status and length of stay were the most frequently reported outcomes. Elevated temperature and fever were associated with poor clinical outcomes in patients with traumatic brain injury, stroke, and cardiac arrest but not in patients with sepsis. Although a causal relationship between elevated temperature and poor outcomes cannot be definitively established, the association observed in this systematic literature review supports the concept that management of elevated temperature may factor in avoidance of detrimental outcomes in multiple critically ill populations. The analysis also highlights gaps in our understanding of fever and elevated temperature in critically ill adult patients.

Quantifying preferences for urea cycle disorder treatments using a discrete choice experiment.

AIMS: Urea cycle disorders (UCDs) can cause ammonia accumulation and central nervous system toxicity. Nitrogen-binding medications can be efficacious, but certain attributes may negatively impact adherence. This study sought to quantify the administration-related attributes influencing overall prescription selection and patient adherence. METHODS: A web-based, quantitative survey including discrete choice experiment (DCE) methodology captured responses from health care providers for patients with UCDs. A series of hypothetical treatment profile sets with attributes such as route of administration, taste/odor, preparation instructions, packaging, dose measurement, and weight use restrictions were presented. From 16 sets of 3 hypothetical product profiles, respondents evaluated attributes most preferred for prescription selection or patient adherence. Attributes assumed a higher overall preference if relative importance (RI) scores were >16.67% (the value if all attributes were of equal importance). Preference weight scores were assessed. A nine-point Likert scale assessed respondent attitudes, such as satisfaction. RESULTS: A total of 51 respondents completed the survey. Respondents reported dissatisfaction with current treatments (mean [SD] = 5.4 [1.7]). For prescription selection, four attributes achieved RI >16.67%: taste/odor (24%), weight restrictions (21%), preparation instructions (18%), and route of administration (17%). For adherence, three attributes related to administration achieved RI >16.67%: taste/odor (28%), preparation instructions (21%), and route of administration (17%). Preference weights for "taste/odor masked" were higher than "not taste/odor masked" for prescription selection (mean [SD]; 1.52 [1.10] vs -1.52 [1.10]) and treatment adherence (73.8 [55.2] vs -73.8 [55.2]). LIMITATIONS: This study contained a relatively small sample size. Survey respondent selection, the use of hypothetical product profiles, and exclusion of non-pharmacologic treatment options could have contributed to potential biases. CONCLUSIONS: Among attributes tested, taste/odor was the most important attribute influencing overall preference for both prescribing and patient adherence, with taste/odor masking preferred. Optimizing nitrogen-binding medications through masking taste/odor may support improved patient adherence and outcomes in UCDs.

A Systematic Literature Review to Assess Fever Management and the Quality of Targeted Temperature Management in Critically Ill Patients.

Targeted temperature management (TTM) has been proposed to reduce mortality and improve neurological outcomes in postcardiac arrest and other critically ill patients. TTM implementation may vary considerably among hospitals, and "high-quality TTM" definitions are inconsistent. This systematic literature review in relevant critical care conditions evaluated the approaches to and definitions of TTM quality with respect to fever prevention and the maintenance of precise temperature control. Current evidence on the quality of fever management associated with TTM in cardiac arrest, traumatic brain injury, stroke, sepsis, and critical care more generally was examined. Searches were conducted in Embase and PubMed (2016 to 2021) following PRISMA guidelines. In total, 37 studies were identified and included, with 35 focusing on postarrest care. Frequently-reported TTM quality outcomes included the number of patients with rebound hyperthermia, deviation from target temperature, post-TTM body temperatures, and number of patients achieving target temperature. Surface and intravascular cooling were used in 13 studies, while one study used surface and extracorporeal cooling and one study used surface cooling and antipyretics. Surface and intravascular methods had comparable rates of achieving target temperature and maintaining temperature. A single study showed that patients with surface cooling had a lower incidence of rebound hyperthermia. This systematic literature review largely identified cardiac arrest literature demonstrating fever prevention with multiple TTM approaches. There was substantial heterogeneity in the definitions and delivery of quality TTM. Further research is required to define quality TTM across multiple elements, including achieving target temperature, maintaining target temperature, and preventing rebound hyperthermia.

Budget impact analysis of belumosudil for chronic graft-versus-host disease treatment in the United States.

AIMS: To assess the impact of belumosudil on the cost of care in chronic graft-versus-host disease (cGVHD) patients who have failed at least two prior lines of systemic therapy using a budget impact model. METHODS: A budget impact model with a 5-year time horizon was constructed in Microsoft Excel. The base case model uses the US prevalence rate of 3 L/4L + cGVHD patients from literature and secondary sources, with the potential for user-defined inputs, including model perspectives. The model includes data for two perspectives: the national US population and a hypothetical US private payer health insurance plan with 10 million (Mil) members. Additional model inputs include market share of cGVHD treatments, their associated adverse event rates, and healthcare resource utilization. RESULTS: The potential annual budget impact for the US national and payer plans was evaluated for cGVHD patients. Based on belumosudil utilization increasing to 55% in 3 L and 4 L + by 2026, cost savings of ∼5.5% and 6.7% ($128.8 and $4.9 Mil USD) were observed from national and payer perspectives, respectively. Cost savings in 2026 were derived from fewer AEs ($108.4 and $3.9 Mil USD, for national and payer perspectives; e.g. neutropenia, and thrombocytopenia) and reduced HCRU ($65.1 and $2.3 Mil USD, for national and payer perspectives; e.g. emergency room visits, ICU stays, etc.). LIMITATIONS: Results from the model were dependent on the available data inputs and assumptions. Real-world values may differ from the assumed performance of treatments, market growth, and treatment dosing and duration. CONCLUSION: The model results suggest that the introduction of belumosudil to treat cGVHD would be associated with substantial cost savings when evaluating a scenario with versus without belumosudil from a US payer perspective.

Clinical and economic impact of primary hyperoxaluria: a retrospective claims analysis.

BACKGROUND: Primary hyperoxaluria (PH) is a family of rare, life-threatening genetic liver disorders characterized by elevated production and excretion of oxalate. To date, the clinical and economic burden associated with PH has not been well characterized due to the rarity of the disease and previous challenges with diagnostic coding that prevented proper identification of patients with PH in claims data. OBJECTIVE: To characterize the clinical and economic costs, as well as health care resource utilization (HCRU), associated with PH relative to a matched cohort of patients without PH. METHODS: Data from the IQVIA PharMetrics Plus Database were used to conduct a retrospective matched-cohort study to compare differences in clinical characteristics, HCRU, and pharmacy and medical costs in patients with PH compared with a matched cohort of patients without PH from January 2014 to December 2019. RESULTS: Overall, 324 patients were included in the PH cohort and 1,620 patients were in the non-PH cohort. The mean age of PH patients was 48.1 years, and approximately 58% of the sample were male. Significantly more patients in the PH cohort than the non-PH cohort were diagnosed with stage 2 chronic kidney disease (CKD; 3.1% vs 0.4%, respectively; P < 0.001), stage 3 CKD (4.6% vs 0.5%; P < 0.001), stage 4 CKD (2.5% vs 0.1%; P < 0.001), and stage 5 CKD or end-stage renal disease (ESRD; 2.2% vs 0.1%; P < 0.001). PH patients had a significantly higher mean Charlson Comorbidity Index composite score than patients in the non-PH cohort (0.79 vs 0.37; P < 0.001). HCRU was significantly higher in patients with PH. The PH cohort had a significantly higher proportion of patients with at least 1 visit to clinicians specializing in nephrology (19% vs 0.4%, respectively; P < 0.001), cardiology (22% vs 12%; P < 0.001), ophthalmology (16% vs 7%; P < 0.001), general surgery (9% vs 6%; P = 0.011), and urology (65% vs 6%; P < 0.001) compared with patients without PH. Mean total annual health care costs in the PH cohort were 65% higher than in the non-PH cohort ($22,549 vs $7,852, respectively; P < 0.001). Similar results were found for total prescription drug costs ($4,125 vs $2,464; P = 0.012). CONCLUSIONS: Despite the rarity of PH, patients with this disease incur substantial clinical and economic burden and may cause financial strain on the health care system. Additional research is warranted to understand the economic and clinical burden of PH stratified by the 3 subtypes of the disease. DISCLOSURES: Funding for this research was provided by Dicerna Pharmaceuticals. Mucha and Hoppe are employed by Dicerna Pharmaceuticals. Silber Miyasoto, Skaar, and Wang are employed by Trinity Life Sciences, which was contracted by Dicerna Pharmaceuticals to conduct the study analysis. Langman is consultant to Dicerna Pharmaceuticals. This study was presented as a poster at the AMCP Nexus 2020 (virtual), October 19-23, 2020, and American Society of Nephrology 2020 (virtual), October 19-25, 2020.

PARC and CUL7 form atypical cullin RING ligase complexes.

CUL7 and the p53-associated, PARkin-like cytoplasmic protein (PARC) were previously reported to form homodimers and heterodimers, the first demonstration of cullin dimerization. Although a CUL7-based SKP1/CUL1/F-box (SCF)-like complex has been observed, little is known about the existence of a PARC-based SCF-like complex and how PARC interacts with CUL7-based complexes. To further characterize PARC-containing complexes, we examined the ability of PARC to form an SCF-like complex. PARC binds RBX1 and is covalently modified by NEDD8, defining PARC as a true cullin. However, PARC fails to bind SKP1 or F-box proteins, including the CUL7-associated FBXW8. To examine the assembly of PARC- and CUL7-containing complexes, tandem affinity purification followed by multidimensional protein identification technology were used. Multidimensional protein identification technology analysis revealed that the CUL7 interaction with FBXW8 was mutually exclusive of CUL7 binding to PARC or p53. Notably, although heterodimers of CUL7 and PARC bind p53, p53 is not required for the dimerization of CUL7 and PARC. The observed physical separation of FBXW8 and PARC is supported functionally by the generation of Parc-/-, Fbxw8-/- mice, which do not show exacerbation of the Fbxw8-/- phenotype. Finally, all of the PARC and CUL7 subcomplexes examined exhibit E3 ubiquitin ligase activity in vitro. Together, these findings indicate that the intricate assembly of PARC- and CUL7-containing complexes is highly regulated, and multiple subcomplexes may exhibit ubiquitin ligase activity.

Dimerization of CUL7 and PARC is not required for all CUL7 functions and mouse development.

CUL7, a recently identified member of the cullin family of E3 ubiquitin ligases, forms a unique SCF-like complex and is required for mouse embryonic development. To further investigate CUL7 function, we sought to identify CUL7 binding proteins. The p53-associated, parkin-like cytoplasmic protein (PARC), a homolog of CUL7, was identified as a CUL7-interacting protein by mass spectrometry. The heterodimerization of PARC and CUL7, as well as homodimerization of PARC and CUL7, was confirmed in vivo. To determine the biological role of PARC by itself and in conjunction with CUL7, a targeted deletion of Parc was created in the mouse. In contrast to the neonatal lethality of the Cul7 knockout mice, Parc knockout mice were born at the expected Mendelian ratios and exhibited no apparent phenotype. Additionally, Parc deletion did not appear to affect the stability or function of p53. These results suggest that PARC and CUL7 form an endogenous complex and that PARC and CUL7 functions are at least partially nonoverlapping. In addition, although PARC and p53 form a complex, the absence of effect of Parc deletion on p53 stability, localization, and function suggests that p53 binding to PARC may serve to control PARC function.

Pulmonary artery catheter use in adult patients undergoing cardiac surgery: a retrospective, cohort study.

BACKGROUND: The utility of pulmonary artery catheters (PACs) and their measurements depend on a variety of factors including data interpretation and personnel training. This US multi-center, retrospective electronic health record (EHR) database analysis was performed to identify associations between PAC use in adult cardiac surgeries and effects on subsequent clinical outcomes. METHODS: This cohort analysis utilized the Cerner Health Facts database to examine patients undergoing isolated coronary artery bypass graft (CABG), isolated valve surgery, aortic surgery, other complex non-valvular and multi-cardiac procedures, and/or heart transplant from January 1, 2011, to June 30, 2015. A total of 6844 adults in two cohorts, each with 3422 patients who underwent a qualifying cardiac procedure with or without the use of a PAC for monitoring purposes, were included. Patients were matched 1:1 using a propensity score based upon the date and type of surgery, hospital demographics, modified European System for Cardiac Operative Risk Evaluation (EuroSCORE II), and patient characteristics. Primary outcomes of 30-day in-hospital mortality, length of stay, cardiopulmonary morbidity, and infectious morbidity were analyzed after risk adjustment for acute physiology score. RESULTS: There was no difference in the 30-day in-hospital mortality rate between treatment groups (OR, 1.17; 95% CI, 0.65-2.10; p = 0.516). PAC use was associated with a decreased length of stay (9.39 days without a PAC vs. 8.56 days with PAC; p < 0.001), a decreased cardiopulmonary morbidity (OR, 0.87; 95% CI, 0.79-0.96; p < 0.001), and an increased infectious morbidity (OR, 1.28; 95% CI, 1.10-1.49; p < 0.001). CONCLUSIONS: Use of a PAC during adult cardiac surgery is associated with decreased length of stay, reduced cardiopulmonary morbidity, and increased infectious morbidity but no increase in the 30-day in-hospital mortality. This suggests an overall potential benefit associated with PAC-based monitoring in this population. TRIAL REGISTRATION: The study was registered at clinicaltrials.gov (NCT02964026) on November 15, 2016.

Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response.

The Integrator complex controls the termination of transcription at diverse classes of gene targets.

Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration targeting-sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3' end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.

SCF ubiquitin ligase-targeted therapies.

The clinical successes of proteasome inhibitors for the treatment of cancer have highlighted the therapeutic potential of targeting this protein degradation system. However, proteasome inhibitors prevent the degradation of numerous proteins, which may cause adverse effects. Increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a multitude of processes at the cellular and organismal levels, and their dysregulation is implicated in many pathologies. SCF ubiquitin ligases are characterized by their high specificity for substrates, and these ligases therefore represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This Review explores and discusses potential strategies to target SCF-mediated biological processes to treat human diseases.

Control of cell growth by the SCF and APC/C ubiquitin ligases.

The ubiquitin-proteasome system plays key roles in the control of cell growth. The cell cycle, in particular, is highly regulated by the functions of the SCF and APC/C ubiquitin ligases, and perturbation of their function can result in tumorigenesis. Although the SCF and APC/C complexes are well established in growth control pathways, many aspects of their function remain unknown. Recent studies have shed light on the mechanism of SCF-mediated ubiquitination and new functions for the SCF complex and APC/C. Our expanding understanding of the roles of the SCF and APC/C complexes highlight the potential for targeted molecular therapies.

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation.

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C(Cdc20) in response to the SAC.