Integrating intracellular and extracellular proteomic profiling for in-depth investigations of cellular communication in a model of prostate cancer.

Cellular communication is essential for cell-cell interactions, maintaining homeostasis and progression of certain disease states. While many studies examine extracellular proteins, the holistic extracellular proteome is often left uncaptured, leaving gaps in our understanding of how all extracellular proteins may impact communication and interaction. We used a cellular-based proteomics approach to more holistically profile both the intracellular and extracellular proteome of prostate cancer. Our workflow was generated in such a manner that multiple experimental conditions can be observed with the opportunity for high throughput integration. Additionally, this workflow is not limited to a proteomic aspect, as metabolomic and lipidomic studies can be integrated for a multi-omics workflow. Our analysis showed coverage of over 8000 proteins while also garnering insights into cellular communication in the context of prostate cancer development and progression. Identified proteins covered a variety of cellular processes and pathways, allowing for the investigation of multiple aspects into cellular biology. This workflow demonstrates advantages for integrating intra- and extracellular proteomic analyses as well as potential for multi-omics researchers. This approach possesses great value for future investigations into the systems biology aspects of disease development and progression.

Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation.

The ubiquity of mass spectrometry-based bottom-up proteomic analyses as a component of biological investigation mandates the validation of methodologies that increase acquisition efficiency, improve sample coverage, and enhance profiling depth. Chromatographic separation is often ignored as an area of potential improvement, with most analyses relying on traditional reversed-phase liquid chromatography (RPLC); this consistent reliance on a single chromatographic paradigm fundamentally limits our view of the observable proteome. Herein, we build upon early reports and validate porous graphitic carbon chromatography (PGC) as a facile means to substantially enhance proteomic coverage without changes to sample preparation, instrument configuration, or acquisition methods. Analysis of offline fractionated cell line digests using both separations revealed an increase in peptide and protein identifications by 43% and 24%, respectively. Increased identifications provided more comprehensive coverage of cellular components and biological processes independent of protein abundance, highlighting the substantial quantity of proteomic information that may go undetected in standard analyses. We further utilize these data to reveal that label-free quantitative analyses using RPLC separations alone may not be reflective of actual protein constituency. Together, these data highlight the value and comprehension offered through PGC-MS proteomic analyses. RAW proteomic data have been uploaded to the MassIVE repository with the primary accession code MSV000091495.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

Complementary proteome and glycoproteome access revealed through comparative analysis of reversed phase and porous graphitic carbon chromatography.

Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes, and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomic experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomic and glycoproteomic datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively.

DDX3X and Stress Granules: Emerging Players in Cancer and Drug Resistance.

The DEAD (Asp-Glu-Ala-Asp)-box helicase 3 X-linked (DDX3X) protein participates in many aspects of mRNA metabolism and stress granule (SG) formation. DDX3X has also been associated with signal transduction and cell cycle regulation that are important in maintaining cellular homeostasis. Malfunctions of DDX3X have been implicated in multiple cancers, including brain cancer, leukemia, prostate cancer, and head and neck cancer. Recently, literature has reported SG-associated cancer drug resistance, which correlates with a negative disease prognosis. Based on the connections between DDX3X, SG formation, and cancer pathology, targeting DDX3X may be a promising direction for cancer therapeutics development. In this review, we describe the biological functions of DDX3X in terms of mRNA metabolism, signal transduction, and cell cycle regulation. Furthermore, we summarize the contributions of DDX3X in SG formation and cellular stress adaptation. Finally, we discuss the relationships of DDX3X, SG, and cancer drug resistance, and discuss the current research progress of several DDX3X inhibitors for cancer treatment.

Age-Dependent Effects of Voluntary Wheel Running Exercise on Voiding Behavior and Potential Age-Related Molecular Mechanisms in Mice.

BACKGROUND: Older men frequently develop lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). Risk factors for LUTS/BPH include sedentary lifestyle, anxiety/depression, obesity, and frailty, which all increase with age. Although physical exercise may reduce the progression and/or severity of LUTS/BPH, the age-related mechanisms responsible remain unknown. METHODS: Voiding symptoms, body mass, and frailty were assessed after 4-weeks of voluntary wheel running in 2-month (n = 10) and 24-month (n = 8) old C57Bl/6J male mice. In addition, various social and individual behaviors were examined in these cohorts. Finally, cellular and molecular markers of inflammation and mitochondrial protein expression were assessed in prostate tissue and systemically. RESULTS: Despite running less (aged vs young X¯ = 12.3 vs 30.6 km/week; p = .04), aged mice had reduced voiding symptoms (X¯ = 67.3 vs 23.7; p < .0001) after 1 week of exercise, which was sustained through week 4 (X¯ = 67.3 vs 21.5; p < .0001). Exercise did not affect voiding symptoms in young mice. Exercise also increased mobility and decreased anxiety in both young and aged mice (p < .05). Exercise decreased expression of a key mitochondrial protein (PINK1; p < .05) and inflammation within the prostate (CD68; p < .05 and plasminogen activator inhibitor-1; p < .05) and in the serum (p < .05). However, a frailty index (X¯ = 0.17 vs 0.15; p = .46) and grip strength (X¯ = 1.10 vs 1.19; p = .24) were unchanged after 4 weeks of exercise in aged mice. CONCLUSIONS: Voluntary aerobic exercise improves voiding behavior and mobility, and decreases prostatic mitochondrial protein expression and inflammation in aged mice. This promising model could be used to evaluate molecular mechanisms of aerobic exercise as a novel lifestyle intervention for older men with LUTS/BPH.

Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes.

Mass spectrometry-based discovery glycoproteomics is highly dependent on the use of chromatography paradigms amenable to analyte retention and separation. When compared against established stationary phases such as reversed-phase and hydrophilic interaction liquid chromatography, reports utilizing porous graphitic carbon have detailed its numerous advantages. Recent efforts have highlighted the utility in porous graphitic carbon in high-throughput glycoproteomics, principally through enhanced profiling depth and liquid-phase resolution at higher column temperatures. However, increasing column temperature has been shown to impart disparaging effects in glycopeptide identification. Herein we further elucidate this trend, describing qualitative and semiquantitative effects of increased column temperature on glycopeptide identification rates, signal intensity, resolution, and spectral count linear response. Through analysis of enriched bovine and human glycopeptides, species with high mannose and sialylated glycans were shown to most significantly benefit and suffer from high column temperatures, respectively. These results provide insight as to how porous graphitic carbon separations may be appropriately leveraged for glycopeptide identification while raising concerns over quantitative and semiquantitative label-free comparisons as the temperature changes. RAW MS glycoproteomic data are available via ProteomeXchange with identifier PXD034354.

Proteome-wide Profiling of Asymmetric Dimethylated Arginine in Human Breast Tumors.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification (PTM) that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine dimethylation (ADMA) is often found in cancer, though little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer. In total, 403 methylated sites from 213 proteins were identified, including 322 novel sites when compared to the PhosphositesPlus database. Moreover, using peptide arrays in vitro, approximately 70% of the putative substrates were validated to be methylated by PRMT1, PRMT4, and PRMT6. Notably, when compared with our previously identified ADMA sites from breast cancer cell lines, only 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource for both PRMT and breast cancer communities for further exploitation of the functions of PRMT dysregulation during breast cancer progression.

SLC13A5/sodium-citrate co-transporter overexpression causes disrupted white matter integrity and an autistic-like phenotype.

Endoplasmic reticulum-based N ɛ-lysine acetylation serves as an important protein quality control system for the secretory pathway. Dysfunctional endoplasmic reticulum-based acetylation, as caused by overexpression of the acetyl coenzyme A transporter AT-1 in the mouse, results in altered glycoprotein flux through the secretory pathway and an autistic-like phenotype. AT-1 works in concert with SLC25A1, the citrate/malate antiporter in the mitochondria, SLC13A5, the plasma membrane sodium/citrate symporter and ATP citrate lyase, the cytosolic enzyme that converts citrate into acetyl coenzyme A. Here, we report that mice with neuron-specific overexpression of SLC13A5 exhibit autistic-like behaviours with a jumping stereotypy. The mice displayed disrupted white matter integrity and altered synaptic structure and function. Analysis of both the proteome and acetyl-proteome revealed unique adaptations in the hippocampus and cortex, highlighting a metabolic response that likely plays an important role in the SLC13A5 neuron transgenic phenotype. Overall, our results support a mechanistic link between aberrant intracellular citrate/acetyl coenzyme A flux and the development of an autistic-like phenotype.

Recent Developments and Applications of Quantitative Proteomics Strategies for High-Throughput Biomolecular Analyses in Cancer Research.

Innovations in medical technology and dedicated focus from the scientific community have inspired numerous treatment strategies for benign and invasive cancers. While these improvements often lend themselves to more positive prognoses and greater patient longevity, means for early detection and severity stratification have failed to keep pace. Detection and validation of cancer-specific biomarkers hinges on the ability to identify subtype-specific phenotypic and proteomic alterations and the systematic screening of diverse patient groups. For this reason, clinical and scientific research settings rely on high throughput and high sensitivity mass spectrometry methods to discover and quantify unique molecular perturbations in cancer patients. Discussed within is an overview of quantitative proteomics strategies and a summary of recent applications that enable revealing potential biomarkers and treatment targets in prostate, ovarian, breast, and pancreatic cancer in a high throughput manner.