Polyphosphate attachment to lysine repeats is a non-covalent protein modification.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.

Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Prophylactic Muscle Free Flap for Total Ankle Arthroplasty With Vascular Risk Factors.

Total ankle arthroplasty (TAA) is a treatment for ankle arthritis that preserves the joint's mobility. Conditions causing poor peripheral blood flow are contraindications for TAA. A 63-year-old man with posttraumatic ankle osteoarthritis who was considered high-risk for TAA due to obesity, history of trauma, tobacco usage, chronic venous stasis, lymphedema, and hypertension subsequently underwent TAA followed by a prophylactic muscle free flap to improve peripheral blood flow and soft tissue integrity. He recovered with no pain and excellent ankle mobility. This case highlights the potential usage of prophylactic muscle free flaps to mitigate vascular risk factors in high-risk patients undergoing TAA.

A Novel Orthoplastic Reconstruction of Relapsed Clubfoot With Total Ankle Arthroplasty.

Congenital clubfoot is addressed in infancy and rarely persists into adulthood. Ankle arthroplasty is an increasingly popular surgical intervention for patients with ankle arthritis since it allows a natural ankle range of motion and completely replaces a degenerative hindfoot. Here, we describe the first successful total ankle arthroplasty (TAA) for a patient with previously treated congenital clubfoot that reverted later in life. To address the patient's poor soft-tissue integument and reduce the likelihood of post-surgical complications, a perioperative latissimus muscle-free flap was performed. This two-staged, novel orthoplastic intervention addressed our patient's ankle issues and appears to be a viable option for clubfoot patients.

Modification of histidine repeat proteins by inorganic polyphosphate.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM), which is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovers 30 human and yeast histidine repeat proteins that undergo histidine polyphosphate modification (HPM). This polyP modification is histidine dependent and non-covalent in nature, although remarkably it withstands harsh denaturing conditions-a hallmark of covalent PTMs. Importantly, we show that HPM disrupts phase separation and the phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting HPM as a potential protein regulatory mechanism.

Proteomics analysis reveals a role for E. coli polyphosphate kinase in membrane structure and polymyxin resistance during starvation.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks. From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Regionally distinct progenitor cells in the lower airway give rise to neuroendocrine and multiciliated cells in the developing human lung.

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

The emerging landscape of eukaryotic polyphosphatases.

Polyphosphate (polyP) is a conserved polymer of inorganic phosphate residues that can reach thousands of moieties in length. PolyP has been implicated in cellular functions ranging from energy and phosphate homeostasis to cell signalling in eukaryotes from yeast to humans. Despite the interest in the role of polyP as a signalling molecule, the spatiotemporal regulation of polyP itself remains poorly understood. This knowledge gap limits our ability to understand how polyP impacts the physiology of normal and diseased cells and how this might be exploited in a therapeutic context. Polyphosphatases, enzymes that degrade polyP to generate shorter chains and free inorganic phosphate are ideally positioned to mediate polyP dynamics. However, little is known about how the activities of these enzymes are linked to specific cellular functions and how they might be regulated. Here, we provide an in-depth overview of polyphosphatase enzymes in budding yeast, which has served as a workhorse for polyP research, and in mammalian cells where the enzymes that make and degrade polyP have remained elusive. We identify critical open questions in both systems and propose strategies to guide future work.

Second-life battery systems for affordable energy access in Kenyan primary schools.

As the world transitions to net zero, energy storage is becoming increasingly important for applications such as electric vehicles, mini-grids, and utility-scale grid stability. The growing demand for storage will constrain raw battery materials, reduce the availability of new batteries, and increase the rate of battery retirement. As retired batteries are difficult to recycle into components, to avoid huge amounts of battery waste, reuse and repurposing options are needed. In this research, we explore the feasibility of using second-life batteries (which have been retired from their first intended life) and solar photovoltaics to provide affordable energy access to primary schools in Kenya. Based on interviews with 12 East African schools, realistic system sizes were determined with varying solar photovoltaic sizes (5-10 kW in 2.5 kW increments) and lithium-ion battery capacities (5-20 kWh in 5 kWh increments). Each combination was simulated under four scenarios as a sensitivity analysis of battery transportation costs (i.e., whether they are sourced locally or imported). A techno-economic analysis is undertaken to compare new and second-life batteries in the resulting 48 system scenarios in terms of cost and performance. We find that second-life batteries decrease the levelized cost of electricity by 5.6-35.3% in 97.2% of scenarios compared to similar systems with new batteries, and by 41.9-64.5% compared to the cost of the same energy service provided by the utility grid. The systems with the smallest levelized cost of electricity (i.e., 0.11 USD/kWh) use either 7.5 kW or 10 kW of solar with 20 kWh of storage. Across all cases, the payback period is decreased by 8.2-42.9% using second-life batteries compared to new batteries; the system with the smallest payback period (i.e., 2.9 years) uses 5 kW solar and 5 kWh storage. These results show second-life batteries to be viable and cost-competitive compared to new batteries for school electrification in Kenya, providing the same benefits while reducing waste.

The Critically Ill Without COVID-19 Infection During the COVID-19 Pandemic: an Analysis of Race and Ethnicity at an Urban Safety-Net Hospital.

PURPOSE: This investigation sought to compare admissions, length of stay, and mortality among medical intensive care unit (MICU) patients without coronavirus disease 2019 (COVID-19) infection admitted to an urban safety-net hospital during the pandemic by patients' self-identified race and ethnicity. MATERIALS AND METHODS: We conducted a retrospective observational study comparing MICU admissions before and during the first surge of COVID-19 illness at an urban, safety-net hospital in Minneapolis, Minnesota. RESULTS: MICU admissions declined from a pre-pandemic average of 968 to 761 during the first COVID surge, including 627 patients (82%) without COVID-19 infection. MICU mortality among patients without COVID-19 infection during the pandemic was 12.8% compared to 9.6% in the pre-pandemic period (p = 0.045). However, rates of non-COVID-19 MICU admissions, mortality, volume, and length of stay did not differ by race and ethnicity between time periods. Of the 131 MICU admissions with COVID-19 infection, patients were more frequently Hispanic ethnicity (24%) or Black (40%), and less frequently White (22%) compared to the pre-pandemic cohort (7%, 30%, and 48%, respectively [p < 0.001]). CONCLUSIONS: During the first COVID-19 surge, MICU admissions for non-COVID-19 disease decreased from pre-pandemic levels, but these patients experienced greater mortality. Unlike critically ill patients admitted with COVID-19 infection, admissions and hospital mortality did not differ by race and ethnicity compared to the pre-pandemic period.

Perioperative changes in left ventricular systolic function following surgical revascularization.

BACKGROUND: Nearly 1/3rd of patients undergoing coronary artery bypass graft surgery (CABG) have left ventricular systolic dysfunction. However, the extent, direction and implications of perioperative changes in left ventricular ejection fraction (LVEF) have not been well characterized in these patients. METHODS: We studied the changes in LVEF among 549 patients with left ventricular systolic dysfunction (LVEF <50%) who underwent CABG as part of the Surgical Treatment for Ischemic Heart Failure (STICH) trial. Patients had pre- and post-CABG (4 month) LVEF assessments using identical cardiac imaging modality, interpreted at a core laboratory. An absolute change of >10% in LVEF was considered clinically significant. RESULTS: Of the 549 patients (mean age 61.4±9.55 years, and 72 [13.1%] women), 145 (26.4%) had a >10% improvement in LVEF, 369 (67.2%) had no change and 35 (6.4%) had >10% worsening of LVEF following CABG. Patients with lower preoperative LVEF were more likely to experience an improvement after CABG (odds ratio 1.36; 95% CI 1.21-1.53; per 5% lower preoperative LVEF; p <0.001). Notably, incidence of postoperative improvement in LVEF was not influenced by presence, nor absence, of myocardial viability (25.5% vs. 28.3% respectively, p = 0.67). After adjusting for age, sex, baseline LVEF, and NYHA Class, a >10% improvement in LVEF after CABG was associated with a 57% lower risk of all-cause mortality (HR: 0.43, 95% CI: 0.26-0.71). CONCLUSIONS: Among patients with ischemic cardiomyopathy undergoing CABG, 26.4% had >10% improvement in LVEF. An improvement in LVEF was more likely in patients with lower preoperative LVEF and was associated with improved long-term survival.

Posttranslational regulation of the GCN5 and PCAF acetyltransferases.

General control nonderepressible 5 protein (Gcn5) and its homologs, including p300/CBP-associated factor (PCAF), are lysine acetyltransferases that modify both histone and non-histone proteins using acetyl coenzyme A as a donor substrate. While decades of studies have uncovered a vast network of cellular processes impacted by these acetyltransferases, including gene transcription and metabolism, far less is known about how these enzymes are themselves regulated. In this review, we summarize the type and functions of posttranslational modifications proposed to control Gcn5 in both yeast and human cells. We further outline common themes, open questions, and strategies to guide future work.

Mortality and readmission risk in relation to QRS duration among patients hospitalized for heart failure with preserved ejection fraction.

BACKGROUND: In ambulatory patients with heart failure (HF) with preserved ejection fraction (HFpEF), QRS prolongation (QRS > 120 msec) and left bundle branch block (LBBB) each carry an increased risk of cardiovascular mortality and/or HF hospitalization. Less is known about implications of conduction abnormalities following an acute HF hospitalization for HFpEF. METHODS AND RESULTS: A retrospective cohort of 1454 patients discharged from after a HF hospitalization between 2015 and 2019 with ejection fraction (EF) ≥ 45% were identified (age 75.1 ± 10.8 years, EF 58.5% ± 10.2%). All patients' electrocardiograms were classified by QRS duration (prolonged - 545 [37.5%] vs. normal [QRS ≤ 120 msec] 909 [62.5%]). QRS prolongation was comprised of: LBBB (4.2%), right bundle branch block (RBBB, 18.3%), intraventricular conduction delay (9.7%), and ventricularly paced (9.7%). Over 4.09 ± 1.00 years, 769 (52.9%) patients died. Survival was similar between normal and prolonged QRS cohorts with an age and sex adjusted hazard ratio of 1.01 (95%CI: 0.87-1.17, p = 0.16). Recurrent HF hospitalization occurred in 91 (16.7%) with QRS prolongation vs. 90 (9.9%) without (odds ratio: 1.82 [95%CI: 1.33-2.50, p < 0.001]). RBBB carried 2.26 higher odds of recurrent HF hospitalization (95%CI: 1.56-3.28). CONCLUSIONS: Following a HF hospitalization, QRS prolongation increased the odds of re-admission for HF in patients with HFpEF without differences in overall mortality.

Ddp1 Cooperates with Ppx1 to Counter a Stress Response Initiated by Nonvacuolar Polyphosphate.

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as by changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level. IMPORTANCE Cells from bacteria to humans have a molecule called polyphosphate (polyP) that functions in diverse processes. In many microbes, polyP is sequestered in granules or lysosome-related organelles such as vacuoles. In this study, we use an ectopic expression system to force budding yeast to accumulate polyP outside the vacuole. We use proteomics to demonstrate that this nonvacuolar polyP initiates a stress response mediated by a signaling cascade involving the Yak1 and Hog1 kinases and the Msn2 and Msn4 transcription factors. This response is countered by a pair of polyphosphatases with different enzymatic activities that function in concert to degrade polyP. Our results provide new insights into why polyP is confined to specific cell locations in many microbial cells.

Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis.

OBJECTIVE: Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease. METHODS: Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model. RESULTS: Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis. CONCLUSIONS: Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.

IRE1α drives lung epithelial progenitor dysfunction to establish a niche for pulmonary fibrosis.

After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase-IRE1α-a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)-or conditional epithelial IRE1α gene knockout-both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvß6, a key activator of transforming growth factor ß (TGF-ß) in pulmonary fibrosis, corresponding to decreased TGF-ß-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19CreERT2-lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.