Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform.

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

Silent information regulator 2 (Sir2) is a conserved NAD+-dependent histone deacetylase crucial for regulating cellular stress response and the aging process in Saccharomyces cerevisiae. In this study, we investigated the molecular mechanism underlying how the absence of Sir2 can lead to altered stress susceptibilities in S. cerevisiae under different environmental and physiological conditions. In a glucose-complex medium, the sir2Δ strain showed increased sensitivity to H2O2 compared to the wild-type strain during the post-diauxic phase. In contrast, it displayed increased resistance during the exponential growth phase. Transcriptome analysis of yeast cells in the post-diauxic phase indicated that the sir2Δ mutant expressed several oxidative defense genes at lower levels than the wild-type, potentially accounting for its increased susceptibility to H2O2. Interestingly, however, the sir2Δras2Δ double mutant exhibited greater resistance to H2O2 than the ras2Δ single mutant counterpart. We found that the expression regulation of the cytoplasmic catalase encoded by CTT1 was critical for the increased resistance to H2O2 in the sir2Δras2Δ strain. The expression of the CTT1 gene was influenced by the combined effect of RAS2 deletion and the transcription factor Azf1, whose level was modulated by Sir2. These findings provide insights into the importance of understanding the intricate interactions among various factors contributing to cellular stress response.

Corrigendum: The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

[This corrects the article DOI: 10.3389/fmicb.2023.1285559.].

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast.

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.

Sir2 phosphorylation through cAMP-PKA and CK2 signaling inhibits the lifespan extension activity of Sir2 in yeast.

Silent information regulator 2 (Sir2), an NAD(+)-dependent protein deacetylase, has been proposed to be a longevity factor that plays important roles in dietary restriction (DR)-mediated lifespan extension. In this study, we show that the Sir2's role for DR-mediated lifespan extension depends on cAMP-PKA and casein kinase 2 (CK2) signaling in yeast. Sir2 partially represses the transcription of lifespan-associated genes, such as PMA1 (encoding an H(+)-ATPase) and many ribosomal protein genes, through deacetylation of Lys 16 of histone H4 in the promoter regions of these genes. This repression is relieved by Sir2 S473 phosphorylation, which is mediated by active cAMP-PKA and CK2 signaling. Moderate DR increases the replicative lifespan of wild-type yeast but has no effect on that of yeast expressing the Sir2-S473E or S473A allele, suggesting that the effect of Sir2 on DR-mediated lifespan extension is negatively regulated by S473 phosphorylation. Our results demonstrate a mechanism by which Sir2 contributes to lifespan extension.

Growth phase-dependent roles of Sir2 in oxidative stress resistance and chronological lifespan in yeast.

Silent Information Regulator 2 (Sir2), a conserved NAD(+)-dependent histone deacetylase, has been implicated as one of the key factors in regulating stress response and longevity. Here, we report that the role of Sir2 in oxidative stress resistance and chronological lifespan is dependent on growth phase in yeast. In exponential phase, sir2Δ cells were more resistant to H2O2 stress and had a longer chronological lifespan than wild type. By contrast, in post-diauxic phase, sir2Δ cells were less resistant to H2O2 stress and had a shorter chronological lifespan than wild type cells. Similarly, the expression of antioxidant genes, which are essential to cope with oxidative stress, was regulated by Sir2 in a growth phase-dependent manner. Collectively, our findings highlight the importance of the metabolic state of the cell in determining whether Sir2 can protect against or accelerate cellular aging of yeast.

Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae.

Vascular endothelial growth factor (VEGF) mediates angiogenesis, which plays a critical role in the development and differentiation of the vascular system. VEGF is a homodimeric glycoprotein that contains one N-glycosylation site. In this study, we evaluated Saccharomyces cerevisiae expression systems producing glycosylated and non-glycosylated splice variants of human VEGF, VEGF121, and VEGF165. The pre region of the mating factor α1 (MFα1) signal sequence was found to perform better than the entire MFα1 prepro signal sequence in secreting glycosylated VEGF. Secretion of non-glycosylated VEGF165 was completely blocked, indicating the importance of glycosylation in VEGF165 secretion. Interestingly, non-glycosylated VEGF165 was secreted when guided by the MFα1 prepro signal sequence, albeit to a lesser degree, compared to glycosylated VEGF165. N-glycosylation in the pro region was required for the prepro sequence to promote VEGF secretion. Furthermore, substitution of asparagine at the VEGF glycosylation site with lysine or glutamic acid increased secretion of non-glycosylated VEGF, a finding not previously reported. Our findings suggest that S. cerevisiae could be a suitable host for secreting biologically active, non-glycosylated VEGF for clinical use.