LHPP Inhibits the Proliferation and Metastasis of Renal Cell Carcinoma

Renal cell carcinoma (RCC) is one of the ten most common cancers in the globe. Despite the diagnosis and treatment of renal cell carcinoma that have made great improvements, the morbidity and mortality rates of renal cell carcinoma remain unchanged remarkably. LHPP is a kind of histidine phosphatases, acting as a tumor suppressor in the progression of various cancers. In this study, we found that LHPP was significantly downregulated in RCC tissues and cell lines. Decreased expression of LHPP was closely correlated with tumor size and postoperative metastasis of RCC patients. In addition, overexpression of LHPP inhibited the proliferation and metastasis of RCC. However, suppression of LHPP promoted the proliferation and metastasis of RCC. In conclusion, our results presented the important role of LHPP in the development and progression of RCC.

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-β-Lactamase Superfamily Domain, Associated with Phytase Activity

ABSTRACTInositol-6-phosphate, also known as phytic acid, is a phosphorus source that plays several important roles in the phosphorus cycle and in cell metabolism. The known characterized enzymes responsible for its degradation, the phytases, are mostly derived from cultured individual microorganisms. The catalytic signatures of phytases are restricted to the molecular domains of four protein superfamilies: histidine phosphatases, protein tyrosine phosphatases, the purple acid phosphatases and the β-propeller phosphatases. During function-based screening of previously generated forest soil metagenomic libraries forEscherichia coliclones conferring phytase activity, two positive clones harboring the plasmids pLP05 and pLP12 were detected. Analysis of the insert sequences revealed the absence of classic phosphatase/phytase signatures of the proteins deduced from the putative genes, but the genesmblp01(pLP05) andmblp02(pLP12) encoded putative metallo-β-lactamases (MBLs). Several MBL representatives are promiscuous proteins with phosphoesterase activity, but phytase activity was previously not reported. Bothmblp01andmblp02were subcloned, expressed, and analyzed. Mblp01 and Mblp02 are members of the lactamase B2 family. Protein modeling showed that the closest structural homologue of both proteins was ZipD ofE. coli. Mblp01 and Mblp02 showed activity toward the majority of the tested phosphorylated substrates, including phytate. The maximal enzyme activities were recorded for Mblp01 at 50°C under acidic conditions and for Mblp02 at 35°C and a neutral pH. In the presence of Cu2+or SDS, the activities of Mblp01 and Mblp02 were strongly inhibited. Analyses of the minimal inhibitory concentrations of several β-lactam antibiotics revealed that recombinantE. colicells carryingmblp01ormblp02showed reduced sensitivity toward β-lactam antibiotics.IMPORTANCEPhytic acid is a phosphorus storage molecule in many plant tissues, a source of phosphorus alternative to phosphate rocks, but it can also be a problematic antinutrient. In comparison to other phosphorus sources, phytic acid exhibits reduced bioavailability. Additionally, it influences functions of secondary messengers and acts as antioxidant in tumor growth prevention. The enzymatic capability to process phytate has been reported for a limited number of protein families. This might be due to the almost exclusive use of proteins derived from individual microorganisms to analyze phytase activity. With such a restriction, the study of the complexity and diversity of the phytases remains incomplete. By using metagenome-derived samples, this study demonstrates the existence of phytase activity in one of the most promiscuous superfamilies, the metallo-β-lactamases. Our results increase the general knowledge on phytase diversity in environmental samples and could provide new avenues for the study and engineering of new biocatalysts.

New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases

Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

Phosphohistidine and phospholysine phosphatase activities in the rat: potential protein-lysine and protein-histidine phosphatases?

We have detected phosphohistidine and phospholysine phosphatase activities in rat tissue extracts using partially phosphorylated, high-molecular-mass (> 10 kDa) polymers of histidine and lysine as substrates. Multiple phosphohistidine- and phospholysine-specific phosphatases were present in these extracts based on observed differences in heat stability, sensitivity to bivalent metal ions and thiol modifying reagents, and/or elution from DE-52 cellulose. The properties of these phosphohistidine and phospholysine phosphatases were distinct from those of the phosphomonoester-specific protein phosphatases or the N-P phosphohydrolases that act on the free phosphoamino acids phosphoarginine, 3-phosphohistidine or phospholysine.