T cell activation triggers reversible inosine-5'-monophosphate dehydrogenase assembly.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants.

Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains.

Kinase Inhibitor Profiling Reveals Unexpected Opportunities to Inhibit Disease-Associated Mutant Kinases.

Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant mutant kinases.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.

A High-Throughput Radiometric Kinase Assay.

Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest.

Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag.

Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag.

Influenza A M2 protein conformation depends on choice of model membrane.

While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein.

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects.

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP) to phosphoinositide-3,4,5-trisphosphate (PIP), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, , has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity.

Explanatory chapter: troubleshooting protein expression: what to do when the protein is not soluble.

Production of soluble protein remains a bottleneck in the biochemistry and structural biology fields. Unfortunately, there is no 'magic bullet' that solves all solubility problems. The following is a protocol to test whether a protein expressed recombinantly is soluble, and possible strategies to circumvent insolubility issues.

Explanatory chapter: troubleshooting recombinant protein expression: general.

One of the most daunting problems for biochemists is the expression of recombinant proteins. Often, the host organism differs from the organism from which the gene coding for the protein of interest was derived. This article provides guidelines to determine whether or not protein expression is a problem, describes possible reasons for low protein expression, and covers several possible solutions.

Gel filtration chromatography (size exclusion chromatography) of proteins.

The protocol described here allows the student to construct a standard curve for a gel filtration column with a separation range of 5-250 kD. The size (hydrodynamic radius) of a protein species stable in a buffer containing Tris-HCl, NaCl, and DTT is determined using this column. Modifications may be made to the buffer to accommodate the protein of interest and the separation range of the column.

Using ion exchange chromatography to purify a recombinantly expressed protein.

Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.

Salting out of proteins using ammonium sulfate precipitation.

Protein solubility is affected by ions. At low ion concentrations (<0.5 M), protein solubility increases along with ionic strength.

A highly selective dual insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) inhibitor derived from an extracellular signal-regulated kinase (ERK) inhibitor.

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation.

A UDP-X diphosphatase from Streptococcus pneumoniae hydrolyzes precursors of peptidoglycan biosynthesis.

The gene for a Nudix enzyme (SP_1669) was found to code for a UDP-X diphosphatase. The SP_1669 gene is localized among genes encoding proteins that participate in cell division in Streptococcus pneumoniae. One of these genes, MurF, encodes an enzyme that catalyzes the last step of the Mur pathway of peptidoglycan biosynthesis.

The human kinome and kinase inhibition.

Protein and lipid kinases play key regulatory roles in a number of biological processes. Unsurprisingly, activating mutations in kinases have been linked to a number of disorders and diseases, most notably cancers. Thus, kinases have emerged as promising clinical targets.

The Nudix hydrolase CDP-chase, a CDP-choline pyrophosphatase, is an asymmetric dimer with two distinct enzymatic activities.

A Nudix enzyme from Bacillus cereus (NCBI RefSeq accession no. NP_831800) catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3'→5' RNA exonuclease activity.

Capitalizing on tumor genotyping: towards the design of mutation specific inhibitors of phosphoinsitide-3-kinase.

PI3Ks catalyze the phosphorylation of the inositol hydroxyls of phosphoinositide membrane components. The changes in phosphorylation of the inositides recruit proteins to the plasma membrane that initiate important signaling cascades. PI3Kα, one of the class IA PI3Ks, is highly mutated in cancers.

The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment.

The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from approximately 15 degrees to 38 degrees .