Rosa Viana

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single‐transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I‐deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser‐46 is assumed to be important for the regulation of sugar metabolism in Gram‐positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser‐46 was either prevented or diminished (replacement of Ser‐46 of HPr with Ala or Thr respectively). In a third mutant, Ile‐47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non‐PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser‐46–Ala or Ile‐47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA‐mediated mechanism of CCR is common to most low G+C Gram‐positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.

Role of AMP-activated protein kinase in autophagy and proteasome function

In this work, we have examined the possible role of AMP-activated protein kinase (a key energy sensor) in regulating intracellular protein degradation. We have found that AICAR, a known activator of AMPK, has a dual effect. On one hand, it inhibits autophagy by a mechanism independent of AMPK activity; AICAR decreases class III PI3-kinase binding to beclin-1 and this effect counteracts and reverses the known positive effect of AMPK activity on autophagy. On the other hand, AICAR inhibits the proteasomal degradation of proteins by an AMPK-dependent mechanism. This is a novel function of AMPK that allows the regulation of proteasomal activity under conditions of energy demand.

A CONSERVED SEQUENCE IMMEDIATELY N-TERMINAL TO THE BATEMAN DOMAINS IN AMP-ACTIVATED PROTEIN KINASE GAMMA SUBUNITS IS REQUIRED FOR THE INTERACTION WITH THE BETA SUBUNITS

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. α, β, and γ, with α being the catalytic subunit and β and γ having regulatory roles. Although several studies have defined different domains in α and β involved in the interaction with the other subunits of the complex, little is known about the regions of the γ subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the γ subunit isoforms and studied the interactions with α and β subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20–25 amino acids in γ1, γ2, and γ3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active αβγ complex. This region is required for the interaction with the β subunits. The interaction between the α and γ subunits does not require this region and occurs instead within the Bateman domains of the γ subunit, although the α-γ interaction does appear to stabilize the β-γ interaction. In addition, sequential deletions from the C termini of the γ subunits indicate that deletion of any of the CBS (cystathionine β-synthase) motifs prevents the formation of a functional complex with the α and β subunits.

TOR kinase pathway and 14-3-3 proteins regulate glucose-induced expression of HXT1, a yeast low-affinity glucose transporter.

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low‐affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14‐3‐3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14‐3‐3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF–Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B‐subunit, in the glucose induction of HXT1 is also discussed. Copyright

Two-hybrid analysis identifies PSMD11, a non-ATPase subunit of the proteasome, as a novel interaction partner of AMP-activated protein kinase

Mammalian AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that acts as a sensor of cellular energy status. It interacts with a great variety of different substrates leading to short-term (i.e. regulation of the activity of different enzymes by direct phosphorylation) and long-term effects (i.e. regulation of transcriptional activity of different transcription factors). In this work, we describe the use of the yeast two-hybrid technology to identify additional proteins that interact with the different subunits of AMPK. We have performed three yeast two-hybrid screenings of a human skeletal muscle cDNA library using three different baits: a constitutively active form of AMPKalpha2 (LexA-AMPKalpha2-T172D) co-expressed with AMPKgamma1, LexA-AMPKbeta2 and LexA-AMPKgamma3. Our results identify novel interaction partners of AMPK in human skeletal muscle. We also further characterize the interaction of AMPK with one of these novel interacting proteins, the non-ATPase subunit of the proteasome PSMD11. Our results indicate that AMPK is able to interact physically with this subunit and modify its phosphorylation status, supporting a possible role for AMPK in regulating proteasome function.

An Esterase Gene from Lactobacillus casei Cotranscribed with Genes Encoding a Phosphoenolpyruvate:Sugar Phosphotransferase System and Regulated by a LevR-Like Activator and σ54 Factor

A new esterase-encoding gene was found in the draft genome sequence of Lactobacillus casei BL23 (CECT5275). It is located in an operon together with genes encoding the EIIA, EIIB, EIIC, and EIID proteins of a mannose class phosphoenolpyruvate:sugar phosphotransferase system. After overproduction in Escherichia coli and purification, the esterase could hydrolyze acetyl sugars, hence the operon was named esu for esterase-sugar uptake genes. Upstream of the genes encoding the EII components (esuABCD) and the esterase (esuE), two genes transcribed in the opposite sense were found which encode a Bacillus subtilis LevR-like transcriptional activator (esuR) and a σ54-like transcriptional factor (rpoN). As compared with the wild-type strain, elevated fructose phosphorylation was detected in L. casei mutants constitutively expressing the esu operon. However, none of the many sugars tested could induce the esu operon. The fact that EsuE exhibits esterase activity on acetyl sugars suggests that this operon could be involved in the uptake and metabolism of esterified sugars. Expression of the esu operon is similar to that of the B. subtilis lev operon: it contains a –12,–24 consensus promoter typical of σ54-regulated genes, and EsuR and RpoN are essential for its transcription which is negatively regulated by EIIBEsu. The esuABCDE transcription unit represents the first σ54-regulated operon in lactobacilli. Furthermore, replacement of His852 in the phosphoenolpyruvate:sugar phosphotransferase system regulation domain II of EsuR with Ala indicated that the transcription activator function of EsuR is inhibited by EIIBEsu-mediated phosphorylation at His852.

Tumor suppressor p27Kip1 undergoes endolysosomal degradation through its interaction with sorting nexin 6

A large body of evidence supports the hypothesis that proteasomal degradation of the growth suppressor p27Kip1 (p27) facilitates mammalian cell cycle progression. However, very few studies have addressed the possibility of proteasome‐independent mechanisms of p27 proteolysis. Here we provide evidence for a novel pathway of p27 degradation via the lysosome that is mediated by its interaction with the endosomal protein sorting nexin 6 (SNX6), a member of the sorting nexin family of vesicular trafficking regulators. p27 and SNX6 interact in vitro and in vivo in mammalian cells, partially colocalize in endosomes, and are present in purified endosomal fractions. Gain‐and loss‐of‐function studies revealed that SNX6 induces endosomal accumulation of p27. Moreover, p27 is detected in lysosomes and inhibition of lysosome‐dependent proteolysis impairs serum‐mediated down‐regulation of p27 in a SNX6‐dependent manner. To validate the localization of p27 in these organelles, we analyzed several cell lines using two different anti‐p27 antibodies, several organelle‐specific markers [e.g., early endosome antigen 1, lysosomal‐associated membrane protein (LAMP) 1, LAMP2, and LysoTracker], and overexpression of fluorescent p27 and SNX6. Remarkably, silencing of SNX6 attenuates p27 down‐regulation in the G1 phase of the mitotic cell cycle and delays cell cycle progression. We therefore conclude that, in addition to the proteasome‐dependent pathway, SNX6‐mediated endolysosomal degradation of p27 also contributes to cell cycle progression in mammalian cells.—Fuster, J. J., González, J. M., Edo, M. D., Viana, R., Boya, P., Cervera, J., Verges, M., Rivera, J., Andrés, V. Tumor suppressor p27Kip1 undergoes endolysosomal degradation through its interaction with sorting nexin 6. FASEB J. 24, 2998–3009 (2010). www.fasebj.org

Selection of Single-Chain Antibodies against the VP8 Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

ABSTRACT Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.

The laforin/malin E3-ubiquitin ligase complex ubiquitinates pyruvate kinase M1/M2

BackgroundLafora disease (LD, OMIM 254780) is a fatal neurodegenerative disorder produced mainly by mutations in two genes: EPM2A, encoding the dual specificity phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. Although it is known that laforin and malin may form a functional complex, the underlying molecular mechanisms of this pathology are still far from being understood.MethodsIn order to gain information about the substrates of the laforin/malin complex, we have carried out a yeast substrate-trapping screening, originally designed to identify substrates of protein tyrosine phosphatases.ResultsOur results identify the two muscular isoforms of pyruvate kinase (PKM1 and PKM2) as novel interaction partners of laforin.ConclusionsWe present evidence indicating that the laforin/malin complex is able to interact with and ubiquitinate both PKM1 and PKM2. This post-translational modification, although it does not affect the catalytic activity of PKM1, it impairs the nuclear localization of PKM2.

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway.