Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and “outside–in” directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.     

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults’ RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.     

Inpatient Perioperative Euglycemic Diabetic Ketoacidosis Due to Sodium-Glucose Cotransporter-2 Inhibitors – Lessons From a Case Series and Strategies to Decrease Incidence

ObjectiveTo identify clinical characteristics and factors associated with the development of euglycemic diabetic ketoacidosis (eDKA), and develop suitable strategies to reduce such events.MethodsElectronic health record (EHR) data were extracted to identify all patients between December 1, 2013, and March 30, 2021, who underwent surgical procedures and had been prescribed a sodium-glucose cotransporter 2 inhibitor (SGLT2i) before these procedures. The resulting list was streamlined to a subset of patients who either had diabetic ketoacidosis (DKA) listed as a hospital diagnosis, postoperative serum bicarbonate ≤ 16 mmol/L, or postoperative serum pH ≤ 7.20. Clinical documentation and laboratory data were reviewed to determine the patients with eDKA.ResultsA total of 2183 procedures conducted on 1307 patients, met the inclusion criteria with the majority (1726, 79.1%) being nonemergent patients. Among 1307 patients, 625 (47.8%) were prescribed empagliflozin, 447 (34.2%) canagliflozin, 214 (16.4%) dapagliflozin, and 21 (1.6%) ertugliflozin, respectively. A total of 8 incidences pertaining to eDKA were noted for 8 unique patients; 5 had undergone emergency surgery whereas 3 had undergone nonemergent procedures. In the 3 nonemergent cases, only 1 patient had received counseling to stop the SGLT2i 3 days before the procedure. In perioperative patients who were prescribed an SGLT2i over 6 years, the incidence of eDKA was 0.17% and 1.1% for nonemergent and emergent procedures, respectively.ConclusionEuglycemic DKA was rare in patients undergoing nonemergent procedures, likely because of preoperative instructions to stop their SGLT2i 3 days before the procedure. Euglycemic DKA was more likely to occur in patients undergoing emergency surgery when the SGLT2i could not be prophylactically stopped.     

Weight-Based Insulin During and After Intravenous Insulin Infusion Reduces Rates of Rebound Hyperglycemia When Transitioning to Subcutaneous Insulin in the Medical Intensive Care Unit

ObjectiveHyperglycemia often occurs after the transition from intravenous insulin infusion (IVII) to subcutaneous insulin. Weight-based basal insulin initiated earlier in the course of IVII in the medical intensive care unit (MICU), and a weight-based basal-bolus regimen after IVII, can potentially improve post-IVII glycemic control by 48 hours.MethodsThis prospective study included 69 patients in MICU who were on IVII for ≥24 hours. Exclusions were end-stage renal disease, type 1 diabetes mellitus, and the active use of vasopressors. The intervention group received weight-based basal insulin (0.2-0.25 units/kg) with IVII and weight-based bolus insulin after IVII. The control group received current care. The primary end points were glucose levels at specific time intervals up to 48 hours after IVII.ResultsThere were 25 patients in the intervention group and 44 in the control group. The mean age of the patients was 59 ± 15 years, 32 (47%) were men, and 52 (78%) had prior diabetes mellitus. The 2 groups were not different (acute kidney injury/chronic kidney disease, pre-existing diabetes mellitus, illness severity, or nothing by mouth status after IVII), except for the steroid use, which was higher in the control group than in the intervention group (34% vs 12%, respectively). Glucose levels were not lower until 36 to 48 hours after IVII (166.8 ± 39.1 mg/dL vs 220.0 ± 82.9 mg/dL, P < .001). When controlling for body mass index, nutritional status, hemoglobin A1C, and steroid use, glucose level was lower starting at 12 to 24 hours out (166.87 mg/dL vs 207.50 mg/dL, P = .015). The frequency of hypoglycemia was similar between the 2 groups (5.0% vs 7.1%). The study did not reach target enrollment.ConclusionThe addition of weight-based basal insulin during, and basal-bolus insulin immediately after, IVII in MICU results in better glycemic control at 24 hours after IVII with no increased hypoglycemia.     

Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

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P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.     

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr^?/?) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr^?/? mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr^?/? compared with 10-week-old Fxr^?/? mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr^?/? mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr^?/?Xbp1^LKO) and Fxr^?/?Xbp1^fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr^?/? mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.     

Transition From Pediatric to Adult Care for Individuals With Type 1 Diabetes: Opportunities and Challenges

ObjectivesType 1 diabetes (T1D) is a chronic disease with patients across the age spectrum that has high potential for morbidity and mortality. Unfortunately, patients transitioning from pediatric to adult care continue to demonstrate worsened glycemic control in part due to lack of understanding of transition of care best practices.MethodsThis review highlights the impact of existing transition of care interventions, assessment tools, and other recently published strategies for providers to consider to improve care of adolescent and young adult (AYA) patients with T1D in both hospital- and clinic-based settings.ResultsMany barriers impact patients with T1D during the transition period and disparities by race, sex, insurance status, and comorbid illness persist. As diabetic care continues to evolve and the prevalence of adolescents and young adults living with T1D increases, an intentional approach to transition of care is more pressing than ever. While current literature on transition of care models is limited, many show promise in improving clinic attendance and decreasing hospitalization. There are critical discussions that providers should lead with AYA patients to improve their outcomes and increase diabetes self-management, such as re-addressing carbohydrate counseling, sleep hygiene, and reproductive planning.ConclusionWhile further research on transition of care is needed, many care models offer the promise of improved T1D outcomes, enhancements in our approach to care, and increased value for our health care system at large.     

SIMSI-Transfer: Software-Assisted Reduction of Missing Values in Phosphoproteomic and Proteomic Isobaric Labeling Data Using Tandem Mass Spectrum Clustering

Isobaric stable isotope labeling techniques such as tandem mass tags (TMTs) have become popular in proteomics because they enable the relative quantification of proteins with high precision from up to 18 samples in a single experiment. While missing values in peptide quantification are rare in a single TMT experiment, they rapidly increase when combining multiple TMT experiments. As the field moves toward analyzing ever higher numbers of samples, tools that reduce missing values also become more important for analyzing TMT datasets. To this end, we developed SIMSI-Transfer (Similarity-based Isobaric Mass Spectra 2 [MS2] Identification Transfer), a software tool that extends our previously developed software MaRaCluster (© Matthew The) by clustering similar tandem MS2 from multiple TMT experiments. SIMSI-Transfer is based on the assumption that similarity-clustered MS2 spectra represent the same peptide. Therefore, peptide identifications made by database searching in one TMT batch can be transferred to another TMT batch in which the same peptide was fragmented but not identified. To assess the validity of this approach, we tested SIMSI-Transfer on masked search engine identification results and recovered >80% of the masked identifications while controlling errors in the transfer procedure to below 1% false discovery rate. Applying SIMSI-Transfer to six published full proteome and phosphoproteome datasets from the Clinical Proteomic Tumor Analysis Consortium led to an increase of 26 to 45% of identified MS2 spectra with TMT quantifications. This significantly decreased the number of missing values across batches and, in turn, increased the number of peptides and proteins identified in all TMT batches by 43 to 56% and 13 to 16%, respectively.     

Reanalysis of ProteomicsDB Using an Accurate,Sensitive, and Scalable False Discovery Rate Estimation Approach for Protein Groups

Estimating false discovery rates (FDRs) of protein identification continues to be an important topic in mass spectrometry–based proteomics, particularly when analyzing very large datasets. One performant method for this purpose is the Picked Protein FDR approach which is based on a target-decoy competition strategy on the protein level that ensures that FDRs scale to large datasets. Here, we present an extension to this method that can also deal with protein groups, that is, proteins that share common peptides such as protein isoforms of the same gene. To obtain well-calibrated FDR estimates that preserve protein identification sensitivity, we introduce two novel ideas. First, the picked group target-decoy and second, the rescued subset grouping strategies. Using entrapment searches and simulated data for validation, we demonstrate that the new Picked Protein Group FDR method produces accurate protein group-level FDR estimates regardless of the size of the data set. The validation analysis also uncovered that applying the commonly used Occam’s razor principle leads to anticonservative FDR estimates for large datasets. This is not the case for the Picked Protein Group FDR method. Reanalysis of deep proteomes of 29 human tissues showed that the new method identified up to 4% more protein groups than MaxQuant. Applying the method to the reanalysis of the entire human section of ProteomicsDB led to the identification of 18,000 protein groups at 1% protein group-level FDR. The analysis also showed that about 1250 genes were represented by ≥2 identified protein groups. To make the method accessible to the proteomics community, we provide a software tool including a graphical user interface that enables merging results from multiple MaxQuant searches into a single list of identified and quantified protein groups.     

Proximity Labeling Facilitates Defining the Proteome Neighborhood of Photosystem II Oxygen Evolution Complex in a Model Cyanobacterium

Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b₆/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.     

The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana

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MS2Rescore: Data-Driven Rescoring Dramatically Boosts Immunopeptide Identification Rates

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Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.