Oncocytic variant of sialadenoma papilliferum - a rare salivary gland tumor: A case report.

Background: Sialadenoma papilliferum (SP) is a rare minor salivary gland neoplasm that accounts for less than 1% of all salivary gland tumors. The tumor typically affects older people, presenting most commonly as a slow-growing tumor of the hard palate, although other anatomical subsites, comprising the oral cavity and parotid glands, have also been reported. Case Report: We report a SP occurring in a 90-year-old female. The patient described feeling a nodule on her palate for several years. The lesion was painless and clinically resembled a round craterlike ulceration of diameter 3 mm. The excisional biopsy was diagnosed histologically as SP. Here, we report the clinicopathological and radiological findings of palatal SP. Conclusions: SP is a rare, benign salivary gland neoplasm, and there are only a few cases described in the literature. Although mostly benign, malignant transformation can occur and should prompt the clinician to ensure complete removal of the tumor tissue. Key words:Sialadenoma papilliferum, minor salivary gland tumor, histopathology, oral pathology, case report.

HPV infection and bacterial microbiota in the semen from healthy men.

BACKGROUND: Aberrant microbiota composition has been linked to disease development at numerous anatomical sites. Microbiota changes in reaction to viral infections, such as human papillomavirus (HPV), have been investigated almost exclusively in the female reproductive tract. However, HPV infection may also affect male health by reducing semen quality and fertility. The aim of this study was to investigate whether present HPV DNA is associated with detectable changes in semen bacterial microbiota composition and diversity. METHODS: This study relied on stored semen samples from 31 fertile healthy men who participated in the Finnish family HPV Study during the years 1998-2001. DNA was extracted from semen with PCR template preparation kit. HPV was genotyped using Luminex-based Multimetrix® assay. Microbiota was analyzed from the V3-V4 region of 16S rDNA gene following sequencing on an Illumina MiSeq platform. All statistical analyses were performed with Calypso software version 8.84. RESULTS: HPV DNA was detected in 19.4% (6/31) of the semen samples. HPV status in the semen did not impact the α-diversity estimations, as measured by Chao1 and Shannon indices, nor ß-diversity. Nevertheless, HPV-positive semen samples exhibited differences in the taxonomic composition of the bacterial microbiota including higher abundances of Moraxellaceae (p = 0.028), Streptococcus (p = 0.0058) and Peptostreptococcus (p = 0.012) compared to HPV-negative semen samples. CONCLUSION: HPV infection is associated with altered bacterial microbiota composition in semen, and this might have in impact to male health in general. As of present, it is unclear whether these changes result from HPV infection or whether altered bacterial microbiota increases susceptibility to HPV infection. More research is needed on viral-bacterial interactions in the male reproductive system.

Oral Microbiota and Cancer Development.

Oral microbiota are among the most diverse in the human body. More than 700 species have been identified in the mouth, and new sequencing methods are allowing us to discover even more species. The anatomy of the oral cavity is different from that of other body sites. The oral cavity has mucosal surfaces (the tongue, the buccal mucosa, the gingiva, and the palate), hard tissues (the teeth), and exocrine gland tissue (major and minor salivary glands), all of which present unique features for microbiota composition. The connection between oral microbiota and diseases of the human body has been under intensive research in the past years. Furthermore, oral microbiota have been associated with cancer development. Patients suffering from periodontitis, a common advanced gingival disease caused by bacterial dysbiosis, have a 2-5 times higher risk of acquiring any cancer compared to healthy individuals. Some oral taxa, especially Porphyromonas gingivalis and Fusobacterium nucleatum, have been shown to have carcinogenic potential by several different mechanisms. They can inhibit apoptosis, activate cell proliferation, promote cellular invasion, induce chronic inflammation, and directly produce carcinogens. These microbiota changes can already be seen with potentially malignant lesions of the oral cavity. The causal relationship between microbiota and cancer is complex. It is difficult to accurately study the impact of specific bacteria on carcinoma development in humans. This review focuses on the elucidating the interactions between oral cavity bacterial microbiota and cancer. We gather literature on the current knowledge of the bacterial contribution to cancer development and the mechanisms behind it.

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells.

BACKGROUND: This study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion. METHODS: HPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1-2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25). RESULTS: All cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki. CONCLUSIONS: Both solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

Composition and maternal origin of the neonatal oral cavity microbiota.

Background: The origin of the initial oral microbiota in neonates still remains poorly understood. Objective: The aim of this study was to understand how the maternal microbiota contributes to the initial neonatal oral microbiota. Design: Twelve mother-neonate pairs with samples from the maternal oral mucosa, uterine cervix and placenta and the neonatal oral cavity immediately after birth were studied. The microbiota composition and diversity were characterized by 16S rRNA gene sequencing (V3-V4 region). The microbiota analyses and comparisons were carried out with Calypso software version 8.1 and with SourceTracker 1.0.1. Results: Samples from the neonatal oral cavity showed moderately high bacterial diversity and low richness. The neonatal oral cavity microbiota seems to share features mainly with the microbes detected in the placenta, followed by the cervical microbiota and the maternal oral microbiota. No statistically significant differences in diversity (Shannon index, p = 0.14), richness (Chao1, p = 0.53) or in microbial composition were observed according to delivery mode. Conclusion: The neonatal oral cavity microbiota is not significantly modulated by the birth canal or maternal oral microbiota but displays clear associations with microbes in the placenta. These results suggest that the neonatal oral microbiota may have a prenatal origin.

HPV infection and bacterial microbiota in breast milk and infant oral mucosa.

OBJECTIVE: We investigated the association between bacterial microbiota in breast milk and the infant mouth. The influence of human papilloma virus (HPV) infection on infant oral microbiota was also assessed. MATERIAL AND METHODS: Altogether 35 breast milk and 35 infant oral samples with known HPV status were selected from the Finnish Family HPV Study cohort. In total, there were 31 mother-infant pairs. The microbiota composition was characterized by 16S rRNA gene sequencing (V3-V4 region). RESULTS: HPV DNA was present in 8.6% (3/35) of the breast milk and 40% (14/35) of the infant oral samples. Eight shared genera between breast milk and infant oral were found; these included Streptococcus, Staphylococcus, Unclassified Gemellaceae, Rothia, Veillonella, Haemophilus, Propionibacterium and Corynebacterium. HPV status was not associated with either microbiota richness or diversity in the infant mouth. However, the infant oral microbiota clustered in different groups according to HPV status. We detected higher abundance of Veillonella dispar (p = 0.048) at species level in HPV negative infant oral samples. We did not detect differences in the breast milk microbiota composition related to HPV infection due to only three HPV positive milk samples. CONCLUSIONS: HPV infection is associated with distinct oral bacterial microbiota composition in infants. The direction of causality underlying the phenomenon remains unclear.

HPV infection and bacterial microbiota in the placenta, uterine cervix and oral mucosa.

We investigated the association between HPV infection and bacterial microbiota composition in the placenta, uterine cervix and mouth in thirty-nine women. HPV DNA genotyping of 24 types was conducted using Multimetrix®. Microbiota composition was characterized by 16S rRNA gene sequencing. HPV DNA was detected in 33% of placenta, 23% cervical and 33% oral samples. HPV16 was the most frequent type in all regions. HPV infection was associated with higher microbiota richness (p = 0.032) in the mouth but did not influence microbial diversity or richness in other samples. HPV infection was associated with higher abundance of Lactobacillaceae (p = 0.0036) and Ureaplasma (LDA score > 4.0, p < 0.05) in the placenta, Haemophilus (p = 0.00058) and Peptostreptococcus (p = 0.0069) genus in the cervix and Selenomonas spp. (p = 0.0032) in the mouth compared to HPV negative samples. These data suggest altered bacterial microbiota composition in HPV positive placenta, cervix and mouth. Whether the changes in bacterial microbiota predispose or result from HPV remains to be determined in future studies.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1ß and interleukin-8.

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1ß. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1ß and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI- mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1ß in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1ß internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Cystathionine ß-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.

Among numerous proteins containing pairs of regulatory cystathionine ß-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.

Cystathionine ß-synthase (CBS) domains confer multiple forms of Mg2+-dependent cooperativity to family II pyrophosphatases.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine ß-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1ß. Some bacterial species can alter their physiological properties as a result of sensing IL-1ß. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1ß sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1ß through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1ß than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1ß, but this binding was not specific, as a control protein for IL-1ß also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1ß-binding surface-exposed lipoprotein that may be part of the bacterial IL-1ß-sensing system.

Interleukin-1ß is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids.

The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1ß, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1ß; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1ß when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1ß and caused IL-1ß leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1ß localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1ß interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1ßin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1ß. The interaction of IL-1ß with conserved HU provides a potential mechanism for shaping bacterial gene expression.

The CBS domain: a protein module with an emerging prominent role in regulation.

Regulatory CBS (cystathionine ß-synthase) domains exist as two or four tandem copies in thousands of cytosolic and membrane-associated proteins from all kingdoms of life. Mutations in the CBS domains of human enzymes and membrane channels are associated with an array of hereditary diseases. Four CBS domains encoded within a single polypeptide or two identical polypeptides (each having a pair of CBS domains at the subunit interface) form a highly conserved disk-like structure. CBS domains act as autoinhibitory regulatory units in some proteins and activate or further inhibit protein function upon binding to adenosine nucleotides (AMP, ADP, ATP, S-adenosyl methionine, NAD, diadenosine polyphosphates). As a result of the differential effects of the nucleotides, CBS domain-containing proteins can sense cell energy levels. Significant conformational changes are induced in CBS domains by bound ligands, highlighting the structural basis for their effects.

The extra-membranous domains of the competence protein HofQ show DNA binding, flexibility and a shared fold with type I KH domains.

Secretins form large oligomeric assemblies in the membrane that control both macromolecular secretion and uptake. Several Pasteurellaceae are naturally competent for transformation, but the mechanism for DNA assimilation is largely unknown. In Haemophilus influenzae, the secretin ComE has been demonstrated to be essential for DNA uptake. In closely related Aggregatibacter actinomycetemcomitans, an opportunistic pathogen in periodontitis, the ComE homolog HofQ is believed to be the outer membrane DNA translocase. Here, we report the structure of the extra-membranous domains of HofQ at 2.3 Å resolution by X-ray crystallography. We also show that the extra-membranous domains of HofQ are capable of DNA binding. The structure reveals two secretin-like folds, the first of which is formed by means of a domain swap. The second domain displays extensive structural similarity to K homology (KH) domains, including the presence of a GxxG motif, which is essential for the nucleotide-binding function of KH domains, suggesting a possible mechanism for DNA binding by HofQ. The data indicate a direct involvement in DNA acquisition and provide insight into the molecular basis for natural competence.

Trimeric form of intracellular ATP synthase subunit ß of Aggregatibacter actinomycetemcomitans binds human interleukin-1ß.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1ß, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1ß receptor has been identified, current knowledge of the bacterial IL-1ß sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1ß than inactive ones. The effect of IL-1ß on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1ß to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1ß, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1ß and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1ß slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1ß. Our results suggest that IL-1ß might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit ß interacted with IL-1ß, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1ß during inflammation.

Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins.

mtCBS-PPase [CBS (cystathionine ß-synthase) domain-containing pyrophosphatase from Moorella thermoacetica] contains a pair of CBS domains that strongly bind adenine nucleotides, thereby regulating enzyme activity. Eight residues associated with the CBS domains of mtCBS-PPase were screened to explore possible associations with regulation of enzyme activity. The majority of the substitutions (V99A, R168A, Y169A, Y169F, Y188A and H189A) enhanced the catalytic activity of mtCBS-PPase, two substitutions (R170A and R187G) decreased activity, and one substitution (K100G) had no effect. AMP-binding affinity was markedly decreased in the V99A, R168A and Y169A mutant proteins, and elevated in the R187G and H189A mutant proteins. Remarkably, the R168A and Y169A substitutions changed the effect of AMP from inhibition to activation. The stoichiometry of AMP binding increased from one to two AMP molecules per CBS domain pair in the Y169F, R170A, R187G and Y188A variants. The ADP-binding affinity decreased in three and increased in four mutant proteins. These findings identify residues determining the strength and selectivity of nucleotide binding, as well as the direction (inhibition or activation) of the subsequent effect. The data suggest that mutations in human CBS domain-containing proteins can be translated into a bacterial context. Furthermore, our data support the hypothesis that the CBS domains act as an 'internal inhibitor' of mtCBS-PPase.

A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides.

CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively.

Characterization and subcellular localization of human neutral class II alpha-mannosidase [corrected].

A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.

A complete structural description of the catalytic cycle of yeast pyrophosphatase.

We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the "closed" wild-type active site, one of the two product phosphates has already dissociated, while the D115E variant "open" conformation is of the hitherto unobserved two-phosphate and two-"bridging" water active site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into the active site. Such subtle changes underline how low the energy barriers are between thermodynamically favorable conformations of the enzyme. The structures also allow us to associate metal binding constants to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the active site, showing for the first time how product is released through a channel of flexible cationic side chains.