Human oral lectin ZG16B acts as a cell wall polysaccharide probe to decode host-microbe interactions with oral commensals.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Functional Specialization of Human Salivary Glands and Origins of Proteins Intrinsic to Human Saliva.

Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract, and they serve as potential diagnostic markers for monitoring human health and disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands and integration with the saliva proteome, the blood plasma proteome, and transcriptomes of 28+ organs, we link human saliva proteins to their source, identify salivary-gland-specific genes, and uncover fetal- and adult-specific gene repertoires. Our results also provide insights into the degree of gene retention during gland maturation and suggest that functional diversity among adult gland types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than by a few gland-specific factors. Finally, we demonstrate the heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as saliva's use as a diagnostic fluid.

Independent amylase gene copy number bursts correlate with dietary preferences in mammals.

The amylase gene (AMY), which codes for a starch-digesting enzyme in animals, underwent several gene copy number gains in humans (Perry et al., 2007), dogs (Axelsson et al., 2013), and mice (Schibler et al., 1982), possibly along with increased starch consumption during the evolution of these species. Here, we present comprehensive evidence for AMY copy number expansions that independently occurred in several mammalian species which consume diets rich in starch. We also provide correlative evidence that AMY gene duplications may be an essential first step for amylase to be expressed in saliva. Our findings underscore the overall importance of gene copy number amplification as a flexible and fast evolutionary mechanism that can independently occur in different branches of the phylogeny.

Proteotoxic stress targeted therapy (PSTT): induction of protein misfolding enhances the antitumor effect of the proteasome inhibitor bortezomib.

Proteotoxic stress (PS) is generated in cells under a variety of conditions involving accumulation of misfolded proteins. To avoid the toxicity of unmitigated PS, cells activate the heat shock response (HSR). HSR involves upregulation of factors such as ubiquitin and the non-housekeeping chaperone Hsp70 which assist with metabolism of aberrant proteins. The PS-HSR axis is a potential anticancer treatment target since many tumor cells display constitutive PS and dependence on HSR due to their rapid rates of proliferation and translation. In fact, induction of PS via stimulation of protein misfolding (hyperthermia), inhibition of proteasomes (bortezomib) or inhibition of Hsp90 (geldanamycin) have all been considered or used for cancer treatment. We found that combination of bortezomib with an inducer of protein misfolding (hyperthermia or puromycin) resulted in enhanced PS. HSR was also induced, but could not mitigate the elevated PS and the cells died via largely p53-independent apoptosis. Thus, combination treatments were more cytotoxic in vitro than the component single treatments. Consistent with this, combination of non-toxic doses of puromycin with bortezomib significantly increased the antitumor activity of bortezomib in a mouse model of multiple myeloma. These results provide support for using combination treatments that disrupt the balance of PS and HSR to increase the therapeutic index of anticancer therapies.

Anti-malaria drug blocks proteotoxic stress response: anti-cancer implications.

The number of physical conditions and chemical agents induce accumulation of misfolded proteins creating proteotoxic stress. This leads to activation of adaptive pro-survival pathway, known as heat shock response (HSR), resulting in expression of additional chaperones. Several cancer treatment approaches, such as proteasome inhibitor Bortezomib and hsp90 inhibitor geldanamycin, involve activation of proteotoxic stress. Low efficacy of these therapies is likely due to the protective effects of HSR induced in treated cells, making this pathway an attractive target for pharmacological suppression. We found that the anti-malaria drugs quinacrine (QC) and emetine prevented HSR in cancer cells, as judged by induction of hsp70 expression. As opposed to emetine, which inhibited general translation, QC did not affect protein synthesis, but rather suppressed inducible HSF1-dependent transcription of the hsp70 gene in a relatively selective manner. The treatment of tumor cells in vitro with a combination of non-toxic concentrations of QC and proteotoxic stress inducers resulted in rapid induction of apoptosis. The effect was similar if QC was substituted by siRNA against hsp70, suggesting that the HSR inhibitory activity of QC was responsible for cell sensitization to proteotoxic stress inducers. QC was also found to enhance the antitumor efficacy of proteotoxic stress inducers in vivo: combinatorial treatment with 17-DMAG + QC resulted in suppression of tumor growth in two mouse syngeneic models. These results reveal that QC is an inhibitor of HSF1-mediated HSR. As such, this compound has significant clinical potential as an adjuvant in therapeutic strategies aimed at exploiting the cytotoxic potential of proteotoxic stress.

Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

Quercetinase pirin makes poliovirus replication resistant to flavonoid quercetin.

Flavonoid quercetin and its derivative, methylquercetin, inhibit the replication of poliovirus in several cell lines. Here, we show that replication of poliovirus is inhibited by quercetin and that the extent of this inhibition depends on the intracellular content of pirin, a quercetinase. HeLa cells contain higher content of pirin protein than normal kidney human epithelial (NKE) or 293 cells do. Poliovirus replication in HeLa cells is significantly more resistant to quercetin than its replication in NKE and 293 cells. Overexpression of pirin reduced antiviral inhibitory effect of quercetin, while siRNA-induced suppression of pirin level made poliovirus replication more sensitive to the flavonoid. The results suggest that quercetinase activity of pirin determines the resistance of poliovirus infection to quercetin.

Serologically defined colon cancer antigen 3 is necessary for the presentation of TNF receptor 1 on cell surface.

Tumor necrosis factor (TNF) induces apoptosis in sensitive cells in culture when used in combination with inhibitors of transcription or translation. We applied the genetic suppressor element (GSE) methodology to search for the genetic elements protecting NIH3T3 cells from TNF-stimulated death. Ten putative GSEs were isolated from TNF-resistant cells, one of which (GSE0-1) corresponded to the cDNA sequence known as the mouse homolog of human serologically defined colon cancer antigen 3 (SDCCAG3). SDCCAG3 protein contains the region similar to the coiled-coil domain of the myosin tail. The same domain is present in the proteins related to the organelles/proteins trafficking, such as kinesin, Golgin-160, and dynein. We proposed that the SDCCAG3 function might be related to protein trafficking and secretion. The expression of the coiledcoil domain as the dominant negative mutant form of SDCCAG3 made the NIH3T3 and HeLa cells resistant to TNF-specific apoptosis. The presentation of TNFR1 at the surface of these cells was reduced, which affected the sensitivity of the cells to the TNF treatment. We recently showed that the inhibition of protein trafficking and secretion depleted the unstable TNFR1 from plasma membrane. The inhibition of SDCCAG3 activity by its dominant negative mutant suppressed the protein trafficking and secretion, and decreased TNFR1 presentation on the cell surface. Based on these results, we presume that SDCCAG3 is important for protein trafficking and presentation of TNFR1 on the cell surface. Therefore, SDCCAG3 can be viewed as a potential target for modulation of TNF response.

Proteolytic cleavage of the p65-RelA subunit of NF-kappaB during poliovirus infection.

Activation of NF-kappaB during viral infection is one of the critical elements in innate immune response. Several virus-specific factors, such as double-stranded RNA, can trigger host defense mechanisms by inducing NF-kappaB-mediated expression of cytokines and interferons. Early stages of poliovirus infection are also associated with degradation of IkappaB alpha and translocation of NF-kappaB into the nucleus. However, at later stages of poliovirus replication the p65-RelA component of the NF-kappaB complex undergoes a specific cleavage that coincides with the onset of intensive poliovirus protein synthesis and the appearance of the activity of poliovirus protease 3C. Indeed, the p65-RelA amino acid sequence contains the recognition site for 3C, and recombinant protein 3C was shown to be capable of proteolytic cleavage of p65-RelA, generating truncated product similar to that observed during poliovirus infection. Cleavage of p65-RelA occurs during replication of ECHO-1 and rhinovirus 14, suggesting that inactivation of NF-kappaB function by proteolytic cleavage of p65-RelA is the common mechanism by which picornaviruses suppress the innate immune response.

The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha.

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.

Dominant negative form of signal-regulatory protein-alpha (SIRPalpha /SHPS-1) inhibits tumor necrosis factor-mediated apoptosis by activation of NF-kappa B.

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.