Kristiina Kanerva

LDL Cholesterol Recycles to the Plasma Membrane via a Rab8a-Myosin5b-Actin-Dependent Membrane Transport Route

Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC- and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a- and Myosin5b (Myo5b)-dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement.

Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1-3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.

Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion.

Background Upon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity. Methodology/Principal Findings Immunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and β-hexosaminidase is not, indicating a granule subtype-specific function for polyamines. Conclusions/Significance The study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.

Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

Brain neurons express ornithine decarboxylase-activating antizyme inhibitor 2 with accumulation in Alzheimer's disease.

Polyamines are small cationic molecules that in adult brain are connected to neuronal signaling by regulating inward‐rectifier K+‐channels and different glutamate receptors. Antizyme inhibitors (AZINs) regulate the cellular uptake of polyamines and activate ornithine decarboxylase (ODC), the rate‐limiting enzyme of polyamine synthesis. Elevated levels of ODC activity and polyamines are detected in various brain disorders including stroke and Alzheimers disease (AD).

Ornithine decarboxylase regulates the activity and localization of rhoA via polyamination

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. Polyamines and ODC are connected to cell proliferation and transformation. Resting cells display a low ODC activity while normal, proliferating cells display fluctuations in ODC activity that coincide with changes in the actin cytoskeleton during the cell cycle. Cancerous cells display constitutively elevated ODC activity. Overexpression of ODC in NIH 3T3 fibroblasts induces a transformed phenotype. The cytoskeletal rearrangements during cytokinesis and cell transformation are intimately coupled to the ODC activity but the molecular mechanisms have remained elusive. In this study we investigated how ODC and polyamines influence the organization of the cytoskeleton. Given that the small G-proteins of the rho family are key modulators of the actin cytoskeleton, we investigated the molecular interactions of rhoA with ODC and polyamines. Our results show that transglutaminase-catalyzed polyamination of rhoA regulates its activity. The polyamination status of rhoA crucially influences the progress of the cell cycle as well as the rate of transformation of rat fibroblasts infected with temperature-sensitive v-src. We also show that ODC influences the intracellular distribution of rhoA. These findings provide novel insights into the mechanisms by which ODC and polyamines regulate the dynamics of the cytoskeleton during cell proliferation and transformation.

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.

Polarized THG Microscopy Identifies Compositionally Different Lipid Droplets in Mammalian Cells

Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.

Severe neurodegenerative disease in brothers with homozygous mutation in POLR1A

In two brothers born to consanguineous parents, we identified an unusual neurological disease that manifested with ataxia, psychomotor retardation, cerebellar and cerebral atrophy, and leukodystrophy. Via linkage analysis and exome sequencing, we identified homozygous c.2801C>T (p.(Ser934Leu)) in POLR1A (encoding RPA194, largest subunit of RNA polymerase I) and c.511C>T (p.(Arg171Trp)) in OSBPL11 (encoding oxysterol-binding protein-like protein 11). Although in silico analysis, histopathologic evidence and functional verification indicated that both variants were deleterious, segregation with the patient phenotype established that the POLR1A defect underlies the disease, as a clinically unaffected sister also was homozygous for the OSBPL11 variant. Decreased nucleolar RPA194 was observed in the skin fibroblasts of only the affected brothers, whereas intracellular cholesterol accumulation was observed in the skin biopsies of the patients and the sister homozygous for the OSBPL11 variant. Our findings provide the first report showing a complex leukodystrophy associated with POLR1A. Variants in three other RNA polymerase subunits, POLR1C, POLR3A and POLR3B, are known to cause recessive leukodystrophy similar to the disease afflicting the present family but with a later onset. Of those, POLR1C is also implicated in a mandibulofacial dysostosis syndrome without leukodystrophy as POLR1A is. This syndrome is absent in the family we present.

The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes.