Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes.

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

Deep proteomic analysis of obstetric antiphospholipid syndrome by DIA-MS of extracellular vesicle enriched fractions.

Proteins in the plasma/serum mirror an individual's physiology. Circulating extracellular vesicles (EVs) proteins constitute a large portion of the plasma/serum proteome. Thus, deep and unbiased proteomic analysis of circulating plasma/serum extracellular vesicles holds promise for discovering disease biomarkers as well as revealing disease mechanisms. We established a workflow for simple, deep, and reproducible proteome analysis of both serum large and small EVs enriched fractions by ultracentrifugation plus 4D-data-independent acquisition mass spectrometry (4D-DIA-MS). In our cohort study of obstetric antiphospholipid syndrome (OAPS), 4270 and 3328 proteins were identified from large and small EVs enriched fractions respectively. Both of them revealed known or new pathways related to OAPS. Increased levels of von Willebrand factor (VWF) and insulin receptor (INSR) were identified as candidate biomarkers, which shed light on hypercoagulability and abnormal insulin signaling in disease progression. Our workflow will significantly promote our understanding of plasma/serum-based disease mechanisms and generate new biomarkers.

Proteomics of Serum Samples for the Exploration of the Pathological Mechanism of Obstetric Antiphospholipid Syndrome.

Obstetric antiphospholipid syndrome (OAPS) is a multisystem disorder characterized by thrombosis or recurrent fetal loss. In this study, we aim to explore the pathological mechanism of OAPS. Herein, we carried out data-independent acquisition (DIA) mass spectrometry quantitative proteomic analysis of serum samples from OAPS patients and healthy controls. A set of 93 differentially expressed proteins was identified, including 75 upregulated and 18 downregulated proteins compared with the levels in controls. Those proteins are enriched in KEGG pathways related to autoimmune diseases, allergic diseases, and pathogen infection. Interestingly, metabolic pathways such as fatty acid degradation and type I diabetes were enriched, indicating that OAPS is metabolic disease related. The significantly increased triglyceride also supported this idea. The differentially expressed proteins insulin-like growth factor-binding protein-1 (IGFBP-1), C-reactive protein (CRP), and ferritin light chain (FTL) were validated by ELISA. Our study presented a deep serum proteomics of OAPS and advanced our understanding of OAPS pathogenesis.

Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2.

In eukaryotes, alternative pre-mRNA splicing allows a single gene to encode different protein isoforms that function in many biological processes, and they are used as biomarkers or therapeutic targets for diseases. Although protein isoforms in the human genome are well annotated, we speculate that some low-abundance protein isoforms may still be under-annotated because most genes have a primary coding product and alternative protein isoforms tend to be under-expressed. A peptide coencoded by a novel exon and an annotated exon separated by an intron is known as a novel junction peptide. In the absence of known transcripts and homologous proteins, traditional whole-genome six-frame translation-based proteogenomics cannot identify novel junction peptides, and it cannot capture novel alternative splice sites. In this article, we first propose a strategy and tool for identifying novel junction peptides, called CJunction, which we then integrate into a proteogenomics process specifically designed for novel protein isoform discovery and apply to the analysis of a deep-coverage HeLa mass spectrometry data set with identifier PXD004452 in ProteomeXchange. We succeeded in identifying and validating three novel protein isoforms of two functionally important genes, NHSL1 (causative gene of Nance-Horan syndrome) and EEF1B2 (translation elongation factor), which validate our hypothesis. These novel protein isoforms have significant sequence differences from the annotated gene-coding products introduced by the novel N-terminal, suggesting that they may play importantly different functions.

CBRPP: a new RNA-centric method to study RNA-protein interactions.

RNA and protein are interconnected biomolecules that can influence each other's life cycles and functions through physical interactions. Abnormal RNA-protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA-protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA-protein complexes manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.

Immune suppression in the early stage of COVID-19 disease.

The outbreak of COVID-19 has become a worldwide pandemic. The pathogenesis of this infectious disease and how it differs from other drivers of pneumonia is unclear. Here we analyze urine samples from COVID-19 infection cases, healthy donors and non-COVID-19 pneumonia cases using quantitative proteomics. The molecular changes suggest that immunosuppression and tight junction impairment occur in the early stage of COVID-19 infection. Further subgrouping of COVID-19 patients into moderate and severe types shows that an activated immune response emerges in severely affected patients. We propose a two-stage mechanism of pathogenesis for this unusual viral infection. Our data advance our understanding of the clinical features of COVID-19 infections and provide a resource for future mechanistic and therapeutics studies.

A study of the functional significance of epidermal growth factor in major depressive disorder.

OBJECTIVE: The precise mechanism of major depressive disorder (MDD) is poorly understood. On the basis of the neurotrophin hypothesis, initial findings from our previous studies, and the functions of epidermal growth factor (EGF) in the central nervous system, we proposed that EGF might contribute to the development of MDD, which was investigated in this study. METHODS: Eight single nucleotide polymorphisms (SNPs) within the EGF gene were genotyped in 463 patients with MDD and 413 control participants among a Chinese population; of these, the plasma EGF levels of 210 patients and 223 controls were determined using the enzyme-linked immunosorbent assay. To determine the effect of functional SNPs on EGF secretion in HEK293 cells, EGF levels in the supernatants of cultured cells were also assessed. RESULTS: None of the informative SNPs showed an allelic association with MDD, but the cis-phase interaction between rs11569017 and rs11569126 was strongly associated with the illness (P=0.0027). The EGF levels in plasma were significantly lower in the patient group than in the control group (P<0.0001). The EGF levels were also significantly lower in patients with the rs11569017-TT genotype than those with either the AA genotype (P=0.013) or the AT genotype (P=0.004); the rs11569017 minor allele significantly affected the expression of the EGF gene (P=0.0004). CONCLUSION: The cis-phase interaction between the SNPs within the EGF locus may contribute toward the etiology of MDD. The plasma EGF levels may be a useful biomarker for the early diagnosis, treatment, and prognosis of major psychiatric disorders.

Association between the epidermal growth factor gene and intelligence in major depression patients.

OBJECTIVE: To study the association between the epidermal growth factor (EGF) gene and intelligence in patients with major depression. METHODS: Intelligence measurement using Wechsler Adult Intelligence Scale (WAIS) was performed on 120 unrelated patients with major depression and 46 control subjects. Blood was collected from all subjects for extraction of genomic DNA. Four single nucleotide polymorphisms (SNPs) in the EGF gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF-MS). RESULTS: Mean scores of both score lang and score task, two subtests in WAIS, differed significantly between major depression patients and controls (P<0.0001). Quantitative trait analysis showed that the genotype of rs2250724 was closely associated with score lang and score task in major depression patients. The associations were still significant after 10 000 permutations. CONCLUSIONS: Although preliminary, our results provide evidence for association between the EGF gene and intelligence in patients with major depression. Genetic variation in the EGF gene may increase the susceptibility of major depression.

Structure of polysaccharides from mycelium and culture medium of Phellinus nigricans using submerged fermentation.

Two water-soluble polysaccharides, PNW1 and PNM1, were respectively isolated from the mycelium and its culture medium of Phellinus nigricans using submerged fermentation before determining their effects on inhibiting the growth of transplantable tumors in mice. The results of the pharmaceutical experiments showed that oral administration of PNW1 and PNM1 (at a dose of 400 mg/kg) inhibited the growth of tumor of mice-transplanted Sarcoma 180 in vivo. Moreover, a higher inhibition ratio of PNW1 (74.70%) was obtained as compared with PNM1 (55.84%). The averaged molecular weight of PNW1 and PNM1 was determined to be 33 and 29 kD, respectively. Both PNW1 and PNM1 were consisted of glucose, galactose, mannose, arabinose and fucose. The major structural features of PNW1 and PNM1 were elucidated using partial acid hydrolysis, periodate oxidation, Smith degradation, 13C-NMR, methylation and GC-MS. On the basis of these results, the repeating units of PNW1 and PNM1 were established.

Anti-tumor and immunomodulating activities of proteoglycans from mycelium of Phellinus nigricans and culture medium.

Two proteoglycans, PNW1 and PNM1, were isolated from the mycelium of Phellinus nigricans through submerged fermentation and culture medium, respectively. PNW1 and PNM1 with similar average molecular weight (33 kDa and 29 kDa) were composed of glucose, galactose, mannose, arabinose and fucose in the molar ratios of 3.26:8.77:6.44:1:1.35 and 20.06:8.72:6.94:1:0.76. At the dose of 100, 200, and 400 mg/kg, PNW1 and PNM1 exhibited anti-tumor activity against mice-transplanted Sarcoma 180 in vivo. However, no direct cytotoxic activity against Sarcoma 180 could be determined. Significant increase in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-alpha) in serum was observed, decreasing the tumor weight significantly. PNW1 and PNM1 could stimulate lymphocytes proliferation and increase production of nitric oxide (NO) and TNF-alpha in macrophages. The results indicate that both lymphocyte and macrophages were activated by preparations of proteoglycans from mycelium and culture medium of P. nigricans. The anti-tumor effect of the proteoglycans is not directly tumoricidal but rather immunostimulating.