Discovering the landscape of protein modifications.

Protein modifications modulate nearly every aspect of cell biology in organisms, ranging from Archaea to Eukaryotes. The earliest evidence of covalent protein modifications was found in the early 20th century by studying the amino acid composition of proteins by chemical hydrolysis. These discoveries challenged what defined a canonical amino acid. The advent and rapid adoption of mass-spectrometry-based proteomics in the latter part of the 20th century enabled a veritable explosion in the number of known protein modifications, with more than 500 discrete modifications counted today. Now, new computational tools in data science, machine learning, and artificial intelligence are poised to allow researchers to make significant progress in discovering new protein modifications and determining their function. In this review, we take an opportunity to revisit the historical discovery of key post-translational modifications, quantify the current landscape of covalent protein adducts, and assess the role that new computational tools will play in the future of this field.

Interpretable multi-omics integration with UMAP embeddings and density-based clustering.

Integrating high-dimensional cellular multi-omics data is crucial for understanding various layers of biological control. Single 'omic methods provide important insights, but often fall short in handling the complex relationships between genes, proteins, metabolites and beyond. Here, we present a novel, non-linear, and unsupervised method called GAUDI (Group Aggregation via UMAP Data Integration) that leverages independent UMAP embeddings for the concurrent analysis of multiple data types. GAUDI uncovers non-linear relationships among different omics data better than several state-of-the-art methods. This approach not only clusters samples by their multi-omic profiles but also identifies latent factors across each omics dataset, thereby enabling interpretation of the underlying features contributing to each cluster. Consequently, GAUDI facilitates more intuitive, interpretable visualizations to identify novel insights and potential biomarkers from a wide range of experimental designs.

The role of calcium-independent phospholipase A2 in cardiolipin remodeling in the spontaneously hypertensive heart failure rat heart.

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)(4)CL]. A decrease in (18:2)(4)CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)(4)CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A(2) (iPLA(2)) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)(4)CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA(2) inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)(4)CL in the failing rat heart, and that mitochondrial iPLA(2) plays a role in the remodeling of CL's acyl composition.

PKC-permitted elevation of sarcolemmal KATP concentration may explain female-specific resistance to myocardial infarction.

The female myocardium, relative to that of the male, exhibits sustained resistance to ischaemic tissue injury, a phenomenon termed sex-specific cardioprotection (SSC). SSC is dependent upon the sarcolemmal K(ATP) channel (sarcK(ATP)), and protein kinase C (PKC). Here we investigate whether PKC-mediated regulation of sarcK(ATP) concentration can explain this endogenous form of protection. Hearts from male (M) and female (F) rats were Langendorff-perfused for 30 min prior to either regional ischaemia-reperfusion (I/R), or global ischaemia (GISC). For both protocols, pre-ischaemic blockade of PKC was achieved by chelerythrine (Chel) in male (M + C) and female (F + C) hearts. Additional female hearts underwent sarcK(ATP) antagonism during I/R by HMR-1098 (HMR), either alone or in combination with Chel (HMR + Chel). GISC hearts were fractionated to assess cellular distribution of PKC and sarcK(ATP). Sex-specific infarct resistance was apparent under control I/R (F, 23 +/- 3% vs. M, 36 +/- 4%, P < 0.05) and abolished by Chel (F + C, 36 +/- 3%). Female infarct resistance was susceptible to sarcK(ATP) blockade (Control, 16 +/- 2% vs. HMR, 27 +/- 3%), and PKC blockade had no additional effect (HMR + Chel, 26 +/- 2%). The prevalence of Kir6.2 and SUR2 was higher in the sarcolemmal fractions of females (Kir6.2: F, 1.24 +/- 0.07 vs. M, 1.02 +/- 0.06; SUR2: F, 3.16 +/- 0.22 vs. M, 2.45 +/- 0.09; ratio units), but normalized by Chel (Kir6.2: F, 1.06 +/- 0.07 vs. M, 0.99 +/- 0.06; SUR2: F, 2.99 +/- 0.09 vs. M, 2.82 +/- 0.22, M; ratio units). Phosphorylation of sarcolemmal PKC was reduced by Chel (p-PKC/PKC: control, 0.43 +/- 0.02; Chel, 0.29 +/- 0.01; P < 0.01). We conclude that PKC-mediated regulation of sarcK(ATP) may account for the physiologically sustainable dependence of SSC upon both PKC and sarcK(ATP), and that this regulation involves PKC-permitted enrichment of the female sarcolemma with sarcK(ATP). As such, the PKC-sarcK(ATP) axis may represent a target for sustainable prophylactic induction of cardioprotection.

A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum.

BACKGROUND: Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum. METHODS: Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins. RESULTS: Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively. CONCLUSIONS: The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.

The tumor suppressor phosphatase PP2A-B56α regulates stemness and promotes the initiation of malignancies in a novel murine model.

Protein phosphatase 2A (PP2A) is a ubiquitously expressed Serine-Threonine phosphatase mediating 30-50% of protein phosphatase activity. PP2A functions as a heterotrimeric complex, with the B subunits directing target specificity to regulate the activity of many key pathways that control cellular phenotypes. PP2A-B56α has been shown to play a tumor suppressor role and to negatively control c-MYC stability and activity. Loss of B56α promotes cellular transformation, likely at least in part through its regulation of c-MYC. Here we report generation of a B56α hypomorph mouse with very low B56α expression that we used to study the physiologic activity of the PP2A-B56α phosphatase. The predominant phenotype we observed in mice with B56α deficiency in the whole body was spontaneous skin lesion formation with hyperproliferation of the epidermis, hair follicles and sebaceous glands. Increased levels of c-MYC phosphorylation on Serine62 and c-MYC activity were observed in the skin lesions of the B56αhm/hm mice. B56α deficiency was found to increase the number of skin stem cells, and consistent with this, papilloma initiation was accelerated in a carcinogenesis model. Further analysis of additional tissues revealed increased inflammation in spleen, liver, lung, and intestinal lymph nodes as well as in the skin lesions, resembling elevated extramedullary hematopoiesis phenotypes in the B56αhm/hm mice. We also observed an increase in the clonogenicity of bone marrow stem cells in B56αhm/hm mice. Overall, this model suggests that B56α is important for stem cells to maintain homeostasis and that B56α loss leading to increased activity of important oncogenes, including c-MYC, can result in aberrant cell growth and increased stem cells that can contribute to the initiation of malignancy.

Endothelial cells mitigate DNA damage and promote the regeneration of hematopoietic stem cells after radiation injury.

Endothelial cells (ECs) are an essential component of the hematopoietic microenvironment, which maintains and regulates hematopoietic stem cells (HSCs). Although ECs can support the regeneration of otherwise lethally-irradiated HSCs, the mechanisms are not well understood. To further understand this phenomenon, we studied HSC regeneration from irradiated bone marrow using co-culture with human aortic ECs (HAECs). Co-culture with HAECs induced a 24-fold expansion of long-term HSCs (CD150(+), lineage(lo), Sca-1(+), c-Kit(+); CD150(+)LSK cells) in vitro. These cells gave rise to functional hematopoietic stem and progenitor cells (HSPCs) with colony-forming activity, multilineage reconstitution and serial transplantation potential. Furthermore, HAECs significantly reduced DNA damage in irradiated LSK cells within 24h. Remarkably, we were able to delay the exposure of irradiated bone marrow to the regenerative, HAEC-derived signals for up to 48h and still rescue functional HSCs. G-CSF is the gold standard for promoting hematopoietic regeneration in vivo. However, when compared to HAECs, in vitro G-CSF treatment promoted lineage differentiation and regenerated 5-fold fewer CD150(+)LSK cells. Together, our results show that HAECs are powerful, direct mitigators of HSC injury and DNA damage. Identification of the HAEC-derived factors that rescue HSCs may lead to improved therapies for hematopoietic regeneration after radiation injury.