Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin ß1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

Critically Ill COVID-19 Patients Exhibit Anti-SARS-CoV-2 Serological Responses.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health care emergency. Anti-SARS-CoV-2 serological profiling of critically ill COVID-19 patients was performed to determine their humoral response. Blood was collected from critically ill ICU patients, either COVID-19 positive (+) or COVID-19 negative (-), to measure anti-SARS-CoV-2 immunoglobulins: IgM; IgA; IgG; and Total Ig (combined IgM/IgA/IgG). Cohorts were similar, with the exception that COVID-19+ patients had a greater body mass indexes, developed bilateral pneumonias more frequently and suffered increased hypoxia when compared to COVID-19- patients (p < 0.05). The mortality rate for COVID-19+ patients was 50%. COVID-19 status could be determined by anti-SARS-CoV-2 serological responses with excellent classification accuracies on ICU day 1 (89%); ICU day 3 (96%); and ICU days 7 and 10 (100%). The importance of each Ig isotype for determining COVID-19 status on combined ICU days 1 and 3 was: Total Ig, 43%; IgM, 27%; IgA, 24% and IgG, 6%. Peak serological responses for each Ig isotype occurred on different ICU days (IgM day 13 > IgA day 17 > IgG persistently increased), with the Total Ig peaking at approximately ICU day 18. Those COVID-19+ patients who died had earlier or similar peaks in IgA and Total Ig in their ICU stay when compared to patients who survived (p < 0.005). Critically ill COVID-19 patients exhibit anti-SARS-CoV-2 serological responses, including those COVID-19 patients who ultimately died, suggesting that blunted serological responses did not contribute to mortality. Serological profiling of critically ill COVID-19 patients may aid disease surveillance, patient cohorting and help guide antibody therapies such as convalescent plasma.

Accurate and reproducible measurements of RhoA activation in small samples of primary cells.

Rho GTPase activation is essential in a wide variety of cellular processes. Measurement of Rho GTPase activation is difficult with limited material, such as tissues or primary cells that exhibit stringent culture requirements for growth and survival. We defined parameters to accurately and reproducibly measure RhoA activation (i.e., RhoA-GTP) in cultured primary keratinocytes in response to serum and growth factor stimulation using enzyme-linked immunosorbent assay (ELISA)-based G-LISA assays. We also established conditions that minimize RhoA-GTP in unstimulated cells without affecting viability, allowing accurate measurements of RhoA activation on stimulation or induction of exogenous GTPase expression.

Nuclear localization of the parafibromin tumor suppressor protein implicated in the hyperparathyroidism-jaw tumor syndrome enhances its proapoptotic function.

Parafibromin is a tumor suppressor protein encoded by HRPT2, a gene recently implicated in the hereditary hyperparathyroidism-jaw tumor syndrome, parathyroid cancer, and a subset of kindreds with familial isolated hyperparathyroidism. Human parafibromin binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex. The mechanism by which loss of parafibromin function can lead to neoplastic transformation is poorly understood. Because the subcellular localization of parafibromin is likely to be critical for its function with the nuclear PAF1 complex, we sought to experimentally define the nuclear localization signal (NLS) of parafibromin and examine its potential role in parafibromin function. Using site-directed mutagenesis, we define a dominant bipartite NLS and a secondary NLS, both in the NH(2)-terminal region of parafibromin whose combined mutation nearly abolishes nuclear targeting. The NLS-mutant parafibromin is significantly impaired in its association with endogenous Paf1 and Leo1. We further report that overexpression of wild-type but not NLS-mutant parafibromin induces apoptosis in transfected cells. Inhibition of endogenous parafibromin expression by RNA interference inhibits the basal rate of apoptosis and apoptosis resulting from DNA damage induced by camptothecin, a topoisomerase I inhibitor. These experiments identify for the first time a proapoptotic activity of endogenous parafibromin likely to be important in its role as a tumor suppressor and show a functional role for the NLS of parafibromin in this activity.

R7-binding protein targets the G protein beta 5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain.

BACKGROUND: Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of G alpha, G beta, and G gamma subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. G beta 5 is the most structurally divergent G beta isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of G beta 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of G beta 5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. RESULTS: We show that endogenous G beta 5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated G beta 5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous G beta 5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed G beta 5/R7-RGS/R7BP proteins. A fraction of endogenous G beta 5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. CONCLUSION: A fraction of G beta 5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed G beta 5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the G beta 5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the G beta 5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex.

Distinction between esterases and lipases: a kinetic study with vinyl esters and TAG.

The better to characterize enzymes hydrolyzing carboxyl ester bonds (carboxyl ester hydrolases), we have compared the kinetic behavior of various lipases and esterases against solutions and emulsions of vinyl esters and TAG. Short-chain vinyl esters are hydrolyzed at comparable rates by esterases and lipases and have higher limits of solubility in water than corresponding TAG. Therefore, they are suited to study the influence of the physical state of the substrate on carboxyl ester hydrolase activity within a large concentration range. Enzymes used in this study are TAG lipases from microorganisms, lipases from human and guinea pig pancreas, pig liver esterase, and acetylcholinesterase. This study also includes cutinase, a fungal enzyme that displays functional properties between esterases and lipases. Esterases display maximal activity against solutions of short-chain vinyl esters (vinyl acetate, vinyl propionate, and vinyl butyrate) and TAG (triacetin, tripropionin, and tributyrin). Half-maximal activity is reached at ester concentrations far below the solubility limit. The transition from solution to emulsion at substrate concentrations exceeding the solubility limit has no effect on esterase activity. Lipases are active on solutions of short-chain vinyl esters and TAG but, in contrast to esterases, they all display maximal activity against emulsified substrates and half-maximal activity is reached at substrate concentrations near the solubility limit of the esters. The kinetics of hydrolysis of soluble substrates by lipases are either hyperbolic or deviate from the Michaelis-Menten model and show no or weak interfacial activation. The presence of molecular aggregates in solutions of short-chain substrates, as evidenced by a spectral dye method, likely accounts for the activity of lipases against soluble esters. Unlike esterases, lipases hydrolyze emulsions of water-insoluble medium- and long-chain vinyl esters and TAG such as vinyl laurate, trioctanoin, and olive oil. In conclusion, comparisons of the kinetic behavior of carboxyl ester hydrolases against solutions and emulsions of vinyl esters and TAG allows the distinction between lipases and esterases. In this respect, it clearly appears that guinea pig pancreatic lipase and cutinase are unambiguously classified as lipases.

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling.

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.