Timothy A. J. Haystead

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Molecular Biologist's Guide to Proteomics

SUMMARY The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems.

Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice

The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). To clarify the physiological roles of ACE2, we generated mice with targeted disruption of the Ace2 gene. ACE2-deficient mice were viable, fertile, and lacked any gross structural abnormalities. We found normal cardiac dimensions and function in ACE2-deficient animals with mixed or inbred genetic backgrounds. On the C57BL/6 background, ACE2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ACE2 had no effect on baseline blood pressures in 129/SvEv mice. After acute Ang II infusion, plasma concentrations of Ang II increased almost 3-fold higher in ACE2-deficient mice than in controls. In a model of Ang II-dependent hypertension, blood pressures were substantially higher in the ACE2-deficient mice than in WT. Severe hypertension in ACE2-deficient mice was associated with exaggerated accumulation of Ang II in the kidney, as determined by MALDI-TOF mass spectrometry. Although the absence of functional ACE2 causes enhanced susceptibility to Ang II-induced hypertension, we found no evidence for a role of ACE2 in the regulation of cardiac structure or function. Our data suggest that ACE2 is a functional component of the renin-angiotensin system, metabolizing Ang II and thereby contributing to regulation of blood pressure.

Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase

ABSTRACT Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression inSaccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by “mixed-peptide” Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.

Getting More from Less Algorithms for Rapid Protein Identification with Multiple Short Peptide Sequences

We describe two novel sequence similarity search algorithms, FASTS and FASTF, that use multiple short peptide sequences to identify homologous sequences in protein or DNA databases. FASTS searches with peptide sequences of unknown order, as obtained by mass spectrometry-based sequencing, evaluating all possible arrangements of the peptides. FASTF searches with mixed peptide sequences, as generated by Edman sequencing of unseparated mixtures of peptides. FASTF deconvolutes the mixture, using a greedy heuristic that allows rapid identification of high scoring alignments while reducing the total number of explored alternatives. Both algorithms use the heuristic FASTA comparison strategy to accelerate the search but use alignment probability, rather than similarity score, as the criterion for alignment optimality. Statistical estimates are calculated using an empirical correction to a theoretical probability. These calculated estimates were accurate within a factor of 10 for FASTS and 1000 for FASTF on our test dataset. FASTS requires only 15–20 total residues in three or four peptides to robustly identify homologues sharing 50% or greater protein sequence identity. FASTF requires about 25% more sequence data than FASTS for equivalent sensitivity, but additional sequence data are usually available from mixed Edman experiments. Thus, both algorithms can identify homologues that diverged 100 to 500 million years ago, allowing proteomic identification from organisms whose genomes have not been sequenced.

Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

The Mammalian Target of Rapamycin Phosphorylates Sites Having a (Ser/Thr)-Pro Motif and Is Activated by Antibodies to a Region near Its COOH Terminus

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [γ-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase.

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

[40] Use of okadaic acid to inhibit protein phosphatases in intact cells

Publisher Summary Okadaic acid is a marine toxin originally isolated from the black sponge, Halichondria okadaii . It is now known to be one of a family of related toxins, including dinophysistoxin 1 and acanthifolicin. These toxins are synthesized by dinoflagellates, especially of the genus Dinophysis , but the toxins accumulate in organisms further up the food chain, including sponges, shellfish, and, ultimately, humans. In humans, consumption of contaminated shellfish causes diarrhetic shellfish poisoning, and this is a particular problem at certain times of year when there is a proliferation of plankton containing the dinoflagellates. Okadaic acid is a complex fatty acid derivative containing numerous polyether linkages. It is an extremely valuable tool for testing the physiological role of protein phosphorylation in any physiological response in intact cells. It has been shown to act as a tumor promoter in the mouse skin bioassay, but unlike many other tumor promoters does not activate protein kinase C. The structure of okadaic acid is very hydrophobic, which makes it possible to be used it as a protein phosphatase inhibitor in intact cells.