PMS2 variant results in loss of ATPase activity without compromising mismatch repair.

Hereditary cancer syndromes account for approximately 5%-10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N-terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in-cell fluorescent-based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign.

Development of a Synthetic 3-ketosteroid Δ1-dehydrogenase for the Generation of a Novel Catabolic Pathway Enabling Cholesterol Degradation in Human Cells.

Cholesterol is an essential component of membranes, which is acquired by cells via receptor-mediated endocytosis of lipoproteins or via de novo synthesis. In specialized cells, anabolic enzymes metabolize cholesterol, generating steroid hormones or bile acids. However, surplus cholesterol cannot be catabolized due to the lack of enzymes capable of degrading the cholestane ring. The inability to degrade cholesterol becomes evident in the development and progression of cardiovascular disease, where the accumulation of cholesterol/cholesteryl-esters in macrophages can elicit a maladaptive immune response leading to the development and progression of atherosclerosis. The discovery of cholesterol catabolic pathways in Actinomycetes led us to the hypothesis that if enzymes enabling cholesterol catabolism could be genetically engineered and introduced into human cells, the atherosclerotic process may be prevented or reversed. Comparison of bacterial enzymes that degrade cholesterol to obtain carbon and generate energy with the action of human enzymes revealed that humans lack a 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), which catalyzes the C-1 and C-2 desaturation of ring A. Here we describe the construction, heterologous expression, and actions of a synthetic humanized Δ1-KstD expressed in Hep3B and U-937 cells, providing proof that one of three key enzymes required for cholesterol ring opening can be functionally expressed in human cells.

The Antitumor Drug LB-100 Is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating with the Active-Site Catalytic Metals in PPP5C.

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene.

Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss-of-function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%-30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42-44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small-angle X-ray scattering, and X-ray crystallography of the N-terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild-type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.

PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors.

Selective inhibitors for each serine/threonine phosphatase (PPP) are essential to investigate the biological actions of PPPs and to guide drug development. Biologically diverse organisms (e.g., cyanobacteria, dinoflagellates, beetles) produce structurally distinct toxins that are catalytic inhibitors of PPPs. However, most toxins exhibit little selectivity, typically inhibiting multiple family members with similar potencies. Thus, the use of these toxins as chemical tools to study the relationship between individual PPPs and their biological substrates, and how disruptions in these relationships contributes to human disease, is severely limited. Here, we show that tautomycetin (TTN) is highly selective for a single PPP, protein phosphatase 1 (PP1/PPP1C). Our structure of the PP1:TTN complex reveals that PP1 selectivity is defined by a covalent bond between TTN and a PP1-specific cysteine residue, Cys127. Together, these data provide key molecular insights needed for the development of novel probes targeting single PPPs, especially PP1.

An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.