PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases, through clinical genetics and gene disruption in mice. Novel, selective PAD4 inhibitors binding to a calcium-deficient form of the PAD4 enzyme have, for the first time, validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation. The therapeutic potential of PAD4 inhibitors can now be explored.

A novel human protein serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively. Their predicted molecular mass is 58 kDa and they comprise a C‐terminal phosphatase catalytic domain and an N‐terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged. The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity). Thus PP5 and PPT1 comprise a new subfamily. The repeats in the N‐terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several proteins that are required for mitosis, transcription and RNA splicing. Bacterially expressed PP5 is able to dephosphorylate serine residues in proteins and is more sensitive than PP1 to the tumour promoter okadaic acid. A 2.3 kb mRNA encoding PP5 is present in all human tissues examined. Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus. This suggests that, like other nuclear TPR‐containing proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.

The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum-and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK!LIMK!cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Tritonextractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

The specificity of the catalytic subunit of protein phosphatase-1 (PP1,) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1,-binding domains on G, and G,, the subunits that target PPI, to hepatic and muscle glycogen, respectively, and on MI,", the subunit that targets PPI, to smooth muscle myosin. G,-(G63 -T93) interacted with PP1, and prevented G, from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate G, from PPI, or affect other characteristic properties of the PPlG, complex. These results indicate that G, contains two PP1,-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, G,-(G63 -N7.5) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates G, from PP1 because phosphate is inserted into the PP1,-binding domain of G,. M,,<)-(Ml -E309) and Ml,,)-(MI -F38), but not Ml,,,-(D39-E309), mimicked the M,,, subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M,,,, subunit and Ml,~l-(MI -E309), neither Ml,,,-(MI -F38) nor M1,,-(D39-E309) suppressed the PPI,-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M,,, subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M,,,-(MI -F38) displaced G, from PPI,, while GM-(G63-T93) displaced M,,,, from PPlC in vitro. These observations indicate that the region(s) of PP1, that interact with G,/G, and MI,, overlap, explaining why different forms of PPI, contain just a single targetting subunit.Keywords: protein phosphatase-1 ; myosin ; smooth muscle ; glycogen metabolism.Protein phosphatase-1 (PPl), one of the major protein serine/ threonine phosphatases of eukaryotic cells, participates in the control of a variety of cellular functions that include glycogen metabolism, muscle contraction, the exit from mitosis (reviewed in [I, 21) and the splicing of mRNA 131. However, evidence has been accumulating that different processes are regulated by ~ Correspondence to P. Cohen, MRC Protein Phosphorylation Unit,

Ah&actThe structures of the Ml,,, and M,, regulatory subunits of protein phosphatase-lM, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N-terminus of the M,,, subunit from rat aorta contains seven ankyrin repeats, while the C-terminus of the M,, subunit from chicken gizzard contains a leucine zipper motif. The M,,e subunit is expressed in two different forms which differ in their C-terminal sequences. One of these is highly homologous to the whole of the M2, subunit.

The ibeA gene (ibe10) previously identified by TnphoA mutagenesis is part of a 50-kDa full-length open-reading frame (ORF) encoded by a 1.37-kb DNA fragment. An isogenic in-frame deletion mutant of ibeA (ZD1) was constructed by chromosomal gene replacement with a suicide plasmid pCVD442 carrying a 2.1-kb DNA fragment with an ibeA deletion. Similar to the previously described TnphoA insertion mutant of ibeA, the isogenic ibeA deletion mutant ZD1 was significantly less invasive in human brain microvascular endothelial cells (BMECs) than the parent strain. The mutant ZD1 was fully complemented by the ibeA ORF. The ibeA gene was subcloned into pET28a(+) and was expressed as a recombinant protein with an N-terminal histidine tag. The recombinant IbeA protein had much greater activity (50 times) in blocking the invasion of BMECs by Escherichia coli K1 than did the partial protein fragment, which provides further evidence that ibeA is an important determinant for E. coli K1 invasion of BMECs.

No abstract

We have previously isolated a form of protein phosphatase-I (PPIM) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PPlC) bound to an M-complex consisting Biochem. 239,. In this paper, we establish that PPIM accounts for nearly all the myosin phosphatase activity in myofibrils, that the M,,,, and M,, subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M,, subunit does not interact with PPlC, but with the C-terminal 72 residues of the M,,,, subunit, a region which is 43% identical to residues 87-161 of the M,, subunit. A fragment of the M,, subunit, M,,-(Ml-L146), which lacks the C-terminal leucine zipper, also bound to the M,,, subunit, but two other fragments M2,-(M1-E110) and M,,-(E110-K186) did not. The MI ,,, and M,, subunits were both found to be myosin-binding proteins. The C-terminal 291 residues of the M,,,, subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M,,,-(MI -E309) and M,,,-(MI-S477) did not. Thus, the region of the M,,,, subunit that stimulates the dephosphorylation of myosin by PPlC is distinct from the region that targets PPlM to myosin. Remarkably, each myosin dimer was capable of binding about 20 mol M,, subunit and many of the M,,-binding sites were located in the myosin rod domain. The potential significance of this observation is discussed.Keywords: protein phosphatase-1 (PPl) ; myosin ; smooth muscle ; muscle contraction.Protein phosphatase-I (PPl), one of the major serinelthreonine-specific protein phosphatases in eukaryotic cells, is regulated by targetting subunits that direct it to particular subcellular loci, modify its substrate specificity, and confer the ability to be regulated by extracellular signals (reviewed in [ 1,21). A significant proportion of the PP1 in vertebrate muscle extracts is associated with myofibrils and has enhanced activity towards the Plight chain of myosin and reduced activity towards other substrates, such as glycogen phosphorylase [3, 41. When isolated from avian (chicken gizzard) [4, 51 or mammalian (pig bladder) [6] smooth muscle, this form of PP1 (PPIM) was found to be composed of three subunits, namely the catalytic subunit of PP1 Note. The novel nucleotide sequence data published here have been deposited with the sequence data banks and are available under accession numbers S74907 and S74622.(PPIC) and two other proteins with molecular masses of 110 kDa and 21 kDa, termed the MI ,,, and M,, subunits, respectively [4, 51. The M,,,, subunit is complexed to both PPlC and the M,, subunit [4], and is the component that modulates the substrate specificity of PPlC because selective removal of the M,, subunit from PPIM does not affect the rate at which either myosin or glycogen phosphorylase are dephosphorylated 171.The MI ,,, subunit has been cloned from rat aorta [5], chicken gizzard [8], and rat kidney [9] cDNA libraries. The N-terminus of the M,,, subunit contains seven ankyrin r...