Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σK

Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σ^K and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σ^K(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.     

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

Background

Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer''s β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

Methodology

Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.

Conclusions

The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer''s disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.     

How Important is Genetics for an Understanding of Evolution?

SYNOPSIS. The contributions of modern genetics to the understandingof evolution have been threefold. First, it has documented theextent of genetic variation that exists in populations as abasis for future evolution. In particular it has shown thatnatural selection has not destroyed all variation, as mightbe predicted, so that there must be mechanisms for the maintenanceandorigin of new selectable variation that balance the homogenizingforces of selection. Second, it has begun to provide a mechanismof the origin of genetic novelties which must be at the basisof the major features of the history of life. In doing so, ithas shown how selection fornew features is at all times historicallycontingent and that evolution is at all times at riskof fallinginto genetic dead-ends. Third, modern genetics has greatly enrichedthe diversity ofmechanisms known to cause evolutionary change.All of these mechanisms involve the conversion of variationbetween individuals into variation between populations in timeand space, but many are non-selective or even counter-selective.Natural selection is not the only mechanism of evolution.     

Structural and Functional Determinants of γ-Secretase,an Intramembrane Protease Implicated in Alzheimer’s Disease

Alzheimer’s disease is the most common form of neurodegenerative diseases in humans, characterized by the progressive accumulation and aggregation of amyloid-β peptides (Aβ) in brain regions subserving memory and cognition. These 39-43 amino acids long peptides are generated by the sequential proteolytic cleavages of the amyloid-β precursor protein (APP) by β- and γ-secretases, with the latter being the founding member of a new class of intramembrane-cleaving proteases (I-CliPs) characterized by their intramembranous catalytic residues hydrolyzing the peptide bonds within the transmembrane regions of their respective substrates. These proteases include the S2P family of metalloproteases, the Rhomboid family of serine proteases, and two aspartyl proteases: the signal peptide peptidase (SPP) and γ-secretase. In sharp contrast to Rhomboid and SPP that function as a single component, γ-secretase is a multi-component protease with complex assembly, maturation and activation processes. Recently, two low-resolution three-dimensional structures of γ-secretase and three high-resolution structures of the GlpG rhomboid protease have been obtained almost simultaneously by different laboratories. Although these proteases are unrelated by sequence or evolution, they seem to share common functional and structural mechanisms explaining how they catalyze intramembrane proteolysis. Indeed, a water-containing chamber in the catalytic cores of both γ-secretase and GlpG rhomboid provides the hydrophilic environment required for proteolysis and a lateral gating mechanism controls substrate access to the active site. The studies that have identified and characterized the structural determinants critical for the assembly and activity of the γ-secretase complex are reviewed here.     

Cleavage of DNA at apyrimidinic sites by lysyl-tryptophyl-α-lysine

Incubation of partially depyrimidinated superhelical or linear DNA with the peptide lysyl-tryptophyl-α-lysine results in specific cleavage of the DNA at the base loss sites. No differences were found between the rates of breakage of apyrimidinic sites and apurinic sites in superhelical DNA.     

Thermodynamic Features of Structural Motifs Formed by β-L-RNA

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by ¹H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.     

Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt_fm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt_fm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt_fm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt_fm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt_fm acylation was followed by rupture of the C⁵–C⁶ bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt_fm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.     

Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition

T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.     

Chemical Cleavage of Bovine β-Lactoglobulin by BNPS-Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine β-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-σK cleavage

Starvation of Bacillus subtilis initiates endosporulation involving formation of mother cell (MC) and forespore (FS) compartments. During engulfment, the MC membrane migrates around the FS and protein channels connect the two compartments. The channels are necessary for postengulfment FS gene expression, which relieves inhibition of SpoIVFB, an intramembrane protease that cleaves Pro-σ^K, releasing σ^K into the MC. SpoIVFB has an ATP-binding domain exposed to the MC cytoplasm, but the role of ATP in regulating Pro-σ^K cleavage has been unclear, as has the impact of the channels on MC and FS ATP levels. Using luciferase produced separately in each compartment to measure relative ATP concentrations during sporulation, we found that the MC ATP concentration rises about twofold coincident with increasing cleavage of Pro-σ^K, and the FS ATP concentration does not decline. Mutants lacking a channel protein or defective in channel protein turnover exhibited modest and varied effects on ATP levels, which suggested that low ATP concentration does not explain the lack of postengulfment FS gene expression in channel mutants. Furthermore, a rise in the MC ATP level was not necessary for Pro-σ^K cleavage by SpoIVFB, based on analysis of mutants that bypass the need for relief of SpoIVFB inhibition.     

Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.     

Rapid process for purification of an extracellular β-xylosidase by aqueous two-phase extraction

A rapid process for purification of an extracellular β-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na₂HPO₄. The aqueous two-phase extraction was performed with PE62 (10%)–NaH₂PO₄ (15%) as phase-forming reagent. SDS–PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.     

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.     

Irradiation as an Alternative to Mastectomy for Early Breast Cancer—An Important Consideration Because of Changes in Laws

Between May 1973 and December 1980 there were 76 patients (78 breasts) with clinical stage I or II breast carcinoma treated by biopsy and definitive radiotherapy at Stanford University Medical Center. Local-regional control has been achieved thus far in 76 of 78 cases (97 percent) with a median follow-up time of 26 months. Transient lymphedema of the breast, arm edema and breast fibrosis were the most commonly noted complications. The cosmetic result was analyzed and scored as excellent in 78 percent, satisfactory in 18 percent and unsatisfactory in 4 percent. The three unsatisfactory results occurred in patients in whom severe fibrosis developed as a result of suboptimal radiation techniques. Interdisciplinary cooperation among surgical, medical and radiation oncologists is important. The 97 percent local-regional control and the 96 percent excellent-to-satisfactory results support the use of primary radiotherapy in early stage breast carcinoma.     

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.