Proteomic Dissection of a Giant Cell

Many individual proteins have been identified as having defined positions relative to cell polarity axes, raising the question of what fraction of all proteins may have polarized localizations. We took advantage of the giant ciliate Stentor coeruleus to quantify the extent of polarized localization proteome-wide. This trumpet-shaped unicellular organism shows a clear morphological anterior-posterior axis defined by a circular array of cilia known as a membranellar band at one end, and a holdfast at the other end. Because individual Stentor cells are over a millimeter in length, we were able to cut the cells into three pieces along the anterior-posterior axis, followed by proteomic analysis of proteins enriched in each piece. We find that approximately 30% of all detected proteins show a polarized location relative to the anterior-posterior cell axis. Proteins with polarized enrichment include centrin-like proteins, calcium-regulated kinases, orthologs of SFI1 and GAS2, and proteases. At the organelle level, nuclear and mitochondrial proteins are enriched in the anterior half of the cell body, but not in the membranellar band itself, while ribosome related proteins are apparently uniformly distributed. RNAi of signaling proteins enriched in the membranellar band, which is the anterior-most structure in the cell, revealed a protein phosphatase 2 subunit b ortholog required for closure of the membranellar band into the ring shape characteristic of Stentor. These results suggest that a large fraction of the Stentor proteome has a polarized localization, and provide a protein-level framework for future analysis of pattern formation and regeneration in Stentor as well as defining a general strategy for subcellular spatial proteomics based on physical dissection of cells.

Differential expression of protein phosphatase 2 scaffold subunit A beta in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding protein phosphatase 2 scaffold subunit A beta, PPP2R1B, when comparing primary tumors of the breast to the tissue of origin, the normal breast. PPP2R1B mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of PPP2R1B in primary tumors of the breast was correlated with recurrence-free survival in patients with luminal A subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. PPP2R1B may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Loss of zinc-finger protein 212 leads to Purkinje cell death and locomotive abnormalities with phospholipase D3 downregulation

Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212–PLD3 relationship is important for Purkinje cell survival.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R'. Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55α mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α role in multiple cellular processes.

PPP2R5D-Related Neurodevelopmental Disorder or Developmental and Epileptic Encephalopathy?: A Novel Phenotypic Description and Review of Published Cases

Background Protein phosphatase 2 regulatory subunit B′ delta (PPP2R5D)-related neurodevelopmental disorder is caused by pathogenic variations in the PPP2R5D gene, product of which is involved in dephosphorylation. This is a rare disorder with description limited to case reports. Its phenotypic spectrum has expanded over the last decade. Methods We report a child with a developmental and epileptic encephalopathy phenotype with a pathogenic PPP2R5D variant. This phenotype has not been previously reported. We also reviewed the previously published reports of patients with this disorder. Results Including the index child, 28 cases (15 girls) were identified from nine relevant research items for analysis. All patients had developmental delay. History of seizures was observed in seven patients while macrocephaly was seen in nearly 80% of patients. Nonneurological manifestations were observed in 13 patients with the most common one being ophthalmological manifestations. The most common genetic variation was c.G592A (p.E198K). The common phenotypic associations of this variation were developmental delay, macrocephaly (11/15), and epilepsy (6/15). Conclusion PPP2R5D gene variations should be suspected in children with developmental delay, autistic features, macrocephaly with or without epilepsy in the absence of any clear etiology. Dysmorphic features might provide a diagnostic clue. DEE phenotype may also be the presenting feature and might be an underreported entity.

Colonic Epithelial PHLPP2 Deficiency Promotes Colonic Epithelial Pyroptosis by Activating the NF-κB Signaling Pathway

Background. Ulcerative colitis (UC) is a chronic inflammatory disease with increasing prevalence worldwide. Barrier defect in intestinal epithelial cells (IECs) is one of the main pathogeneses in UC. Pyroptosis is a programmed lytic cell death and is triggered by inflammatory caspases, while little is known about its role in UC. Methods. Differentially expressed genes (DEGs) were identified by comparing UC patients with healthy controls from the GEO datasets. The candidate genes involved in pyroptosis were obtained, and the underlying molecular mechanism in the progression of UC was explored in vivo and in vitro. Results. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2), a protein phosphatase, was downregulated and involved in regulating inflammation-induced IEC pyroptosis by modulating the NF-κB signaling in UC through bioinformatics analysis. Moreover, we demonstrated that PHLPP2 was downregulated in UC patients and UC mice. Besides, we found that PHLPP2 depletion activated the NF-κB signaling and increased the expressions of caspase-1 P20, Gasdermin N, IL-18, and IL-1β contributing to IEC pyroptosis and inflammation in UC mice. Furthermore, we found that PHLPP2-/- mice developed hypersensitivity to dextran sulfate sodium (DSS) treatment toward colitis showing activated NF-κB signaling and dramatically induced expressions of caspase-1 P20, Gasdermin N, IL-18, and IL-1β. Mechanically, this inflammation-induced downregulation of PHLPP2 was alleviated by an NF-κB signaling inhibitor in intestinal organoids of PHLPP2-/- mice and fetal colonic cells. Conclusions. PHLPP2 downexpression activated the NF-κB signaling and promoted the IEC pyroptosis, leading to UC progression. Therefore, PHLPP2 might be an attractive candidate therapeutic target for UC.

LINC00174 Facilitates Cell Proliferation, Cell Migration and Tumor Growth of Osteosarcoma via Regulating the TGF-β/SMAD Signaling Pathway and Upregulating SSH2 Expression

Osteosarcoma (OS), a frequent malignant tumor which mainly occurs in the bone. The roles of long noncoding RNAs (lncRNAs) have been revealed in cancers, including OS. LncRNA long intergenic non-protein coding RNA (LINC00174) has been validated as an oncogene in several cancers. However, the role of LINC00174 in OS has not been explored. In our research, loss-of-function assays were conducted to explore the function of LINC00174 in OS cells. Then, we explored the downstream pathway of LINC00174 in OS cells. Bioinformatics, RNA pull-down and RIP experiments investigated the downstream mechanism of LINC00174 in OS cells. Finally, in vivo assays clarified the effect of LINC00174 on tumorigenesis. We found that LINC00174 was upregulated in OS tissues and cells. LINC00174 knockdown repressed OS cell growth. Mechanistically, LINC00174 knockdown suppressed the TGF-β/SMAD pathway. LINC00174 interacted with miR-378a-3p, and slingshot protein phosphatase 2 (SSH2) 3′UTR was targeted by miR-378a-3p in OS cells. Rescue assays showed that SSH2 upregulation or miR-378a-3p inhibition counteracted the inhibitory effect of LINC00174 depletion in OS cell growth. Additionally, LINC00174 depletion suppressed tumor growth in mice. In conclusion, LINC00174 promotes OS cellular malignancy and tumorigenesis via the miR-378a-3p/SSH2 axis and the TGF-β/SMAD pathway, which might provide a novel insight for OS treatment.

Proteome Analysis of USP7 Substrates Revealed Its Role in Melanoma Through PI3K/Akt/FOXO and AMPK Pathways

The ubiquitin-specific protease 7 (USP7), as a deubiquitinating enzyme, plays an important role in tumor progression by various mechanisms and serves as a potential therapeutic target. However, the functional role of USP7 in melanoma remains elusive. Here, we found that USP7 is overexpressed in human melanoma by tissue microarray. We performed TMT-based quantitative proteomic analysis to evaluate the A375 human melanoma cells treated with siRNA of USP7. Our data revealed specific proteins as well as multiple pathways and processes that are impacted by USP7. We found that the phosphatidylinositol-3-kinases/Akt (PI3K-Akt), forkhead box O (FOXO), and AMP-activated protein kinase (AMPK) signaling pathways may be closely related to USP7 expression in melanoma. Moreover, knockdown of USP7 in A375 cells, particularly USP7 knockout using CRISPR-Cas9, verified that USP7 regulates cell proliferation in vivo and in vitro. The results showed that inhibition of USP7 increases expression of the AMPK beta (PRKAB1), caspase 7(CASP7), and protein phosphatase 2 subunit B R3 isoform (PPP2R3A), while attenuating expression of C subunit of vacuolar ATPase (ATP6V0C), and peroxisomal biogenesis factor 11 beta (PEX11B). In summary, these findings reveal an important role of USP7 in regulating melanoma progression via PI3K/Akt/FOXO and AMPK signaling pathways and implicate USP7 as an attractive anticancer target for melanoma.