Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (∼100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.IDE³ is an ∼110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (∼100 nm) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-β (Aβ), a peptide vital to the progression of Alzheimer disease (7, 8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of Aβ (5, 9–12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter- and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the disulfide bonds of insulin. Structural analysis reveals that human IDE contains a catalytic chamber formed by the internal cavity of two roughly equally sized ∼55-kDa N- and C-terminal halves (IDE-N and IDE-C, respectively) (2). Within this chamber, only one catalytic center exists. However, IDE cleaves insulin at multiple sites on both the insulin A and B chains to completely inactivate this hormone. It remains unclear whether the cleavages of insulin by IDE proceed in a sequential or stochastic manner.IDE represents an emerging protease family that utilizes an enclosed catalytic chamber to selectively recognize and unfold the substrates for their degradation (1). The volume of the enclosed chamber of IDE (∼16,000 ų) allows the preferential exclusion of peptides that are greater than ∼75 amino acids long. This chamber also has unique electrostatic properties; the internal cavity of IDE-N is predominantly negative, whereas that of IDE-C is positive. Inside the catalytic chamber, IDE has an exosite that is an evolutionarily conserved substrate-binding site ∼30 Å away from the catalytic groove. This exosite is used to anchor the N-terminal end of IDE substrates. The unique size, electrostatic potential, and exosite of Ides'' catalytic chamber are postulated as key factors for the selective binding and unfolding of IDE substrates (1, 2, 14). In addition, one common feature among the known IDE substrates is their higher propensity to form amyloid fibers (8). Amyloidogenic peptides tend to unfold by themselves, which could facilitate their unfolding and subsequent cleavage by IDE. However, the molecular basis of how the catalytic chamber of IDE binds, unfolds, and cleaves insulin into pieces and how the flexibility of this substrate contributes to its cleavage by IDE remain elusive.IDE is known to cut insulin at multiple sites, and the resulting cleavage products are quite complex (4, 15–18). Here we took advantage of the high mass accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and the selective targeting of disulfide bonds by electron capture dissociation (ECD) in our mass spectrometry (MS) analysis to unambiguously identify IDE-degraded fragments of human insulin, as well as the time-dependent production of these fragments. We also present a 2.6- Å insulin-bound IDE structure, revealing extensive shape and charge complementarity of the partially unfolded insulin with the enclosed catalytic chamber and a potential path for the unfolding of insulin. Together, our data elucidate the molecular basis by which IDE engulfs, unfolds, and effectively cleaves insulin into pieces.     

Screening and Evaluation of Human Intestinal Lactobacilli for the Development of Novel Gastrointestinal Probiotics

The aim of this study was to screen intestinal lactobacilli strains for their advantageous properties to select those that could be used for the development of novel gastrointestinal probiotics. Ninety-three isolates were subjected to screening procedures. Fifty-nine percent of the examined lactobacilli showed the ability to auto-aggregate, 97% tolerated a high concentration of bile (2% w/v), 50% survived for 4 h at pH 3.0, and all strains were unaffected by a high concentration of pancreatin (0.5% w/v). One Lactobacillus buchneri strain was resistant to tetracycline. None of the tested strains caused lysis of human erythrocytes. Six potential probiotic strains were selected for safety evaluation in a mouse model. Five of 6 strains caused no translocation, and were considered safe. In conclusion, several strains belonging to different species and fermentation groups were found that have properties required for a potential probiotic strain. This study was the first phase of a multi-phase study aimed to develop a novel, safe and efficient prophylactic and therapeutic treatment system against gastrointestinal infections using genetically modified probiotic lactobacilli.     

The seed dispersal catapult of Cardamine parviflora (Brassicaceae) is efficient but unreliable

? Premise of the study: Seed dispersal performance is an essential component of plant fitness. Despite their significance in shaping performance, the mechanical processes that drive dispersal are poorly understood. We have quantified seed dispersal mechanics in Cardamine parviflora (Brassicaceae), a ballistic disperser that launches seeds with specialized catapult-like structures. To determine which aspects of catapult function dictate interspecific dispersal differences, we compared this disperser with other ballistic dispersers. Comparison with brassicas that lack ballistic dispersal may also provide insight into the evolution of this mechanism. ? Methods: Catapult performance was quantified using high-speed video analysis of dehiscence, ballistic modeling of seed trajectories, and measuring the mechanical energy storage capacity of the spring-like siliqua valve tissue that launched the seeds. ? Key results: The siliquae valves coiled rapidly outward, launching the seeds in 4.7 ± 1.3 ms (mean ± SD, N = 11). Coiling was likely driven by the bilayered valve structure. The catapult was 21.3 ± 10.3% efficient (mean ± SD, N = 11) at transferring stored elastic energy to the seeds as kinetic energy. The majority of seeds (71.4%) were not launched effectively. ? Conclusions: The efficiency of the C. parviflora catapult was high in comparison to that of a ballistic diplochore, a dispersal mode associated with poor ballistic performance, although the unreliability of the launch mechanism limited dispersal distance. Effective launching requires temporary seed-valve adhesion. The adhesion mechanism may be the source of the unreliability. Valve curvature is likely driven by the bilayered valve structure, a feature absent in nonballistic brassicas.     

Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes: CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS*?

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.     

Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803

Searching for enzymes and other proteins which can be redox-regulated by dithiol/disulphide exchange is a rapidly expanding area of functional proteomics. Recently, several experimental approaches using thioredoxins have been developed for this purpose. Thioredoxins comprise a large family of redox-active enzymes capable of reducing protein disulphides to cysteines and of participating in a variety of processes, such as enzyme modulation, donation of reducing equivalents and signal transduction. In this study we screened the target proteomes of three different thioredoxins from the unicellular cyanobacterium Synechocystis sp. PCC 6803, using site-directed active-site cysteine-to-serine mutants of its m-, x- and y-type thioredoxins. The properties of a thioredoxin that determine the outcome of such analyses were found to be target-binding capacity, solubility and the presence of non-active-site cysteines. Thus, we explored how the choice of thioredoxin affects the target proteomes and we conclude that the m-type thioredoxin, TrxA, is by far the most useful for screening of disulphide proteomes. Furthermore, we improved the resolution of target proteins on non-reducing/reducing 2-DE, leading to the identification of 14 new potentially redox-regulated proteins in this organism. The presence of glycogen phosphorylase among the newly identified targets suggests that glycogen breakdown is redox-regulated in addition to glycogen synthesis.     

Histone deacetylase inhibitors (HDI) cause DNA damage in leukemia cells: a mechanism for leukemia-specific HDI-dependent apoptosis?

Histone deacetylase inhibitors (HDI) increase gene expression through induction of histone acetylation. However, it remains unclear whether increases in specific gene expression events determine the apoptotic response following HDI administration. Herein, we show that a variety of HDI trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses but also actual DNA damage, which is significantly increased in leukemic cells compared with normal cells. Thus, increase in H2AX and ataxia telangiectasia mutated (ATM) phosphorylation, early markers of DNA damage, occurs rapidly following HDI administration. Activation of the DNA damage and repair response following HDI treatment is further emphasized by localizing DNA repair proteins to regions of DNA damage. These events are followed by subsequent apoptosis of neoplastic cells but not normal cells. Our data indicate that induction of apoptosis by HDI may result predominantly through accumulation of excessive DNA damage in leukemia cells, leading to activation of apoptosis.     

Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome

We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.     

Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.     

The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo

The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.