Smooth Functional Transition along a Mutational Pathway with an Abrupt Protein Fold Switch

Recent protein design experiments have demonstrated that proteins can migrate between folds through the accumulation of substitution mutations without visiting disordered or nonfunctional points in sequence space. To explore the biophysical mechanism underlying such transitions we use a three-letter continuous protein model with seven atoms per amino acid to provide realistic sequence-structure and sequence-function mappings through explicit simulation of the folding and interaction of model sequences. We start from two 16-amino-acid sequences folding into an α-helix and a β-hairpin, respectively, each of which has a preferred binding partner with 35 amino acids. We identify a mutational pathway between the two folds, which features a sharp fold switch. By contrast, we find that the transition in function is smooth. Moreover, the switch in preferred binding partner does not coincide with the fold switch. Discovery of new folds in evolution might therefore be facilitated by following fitness slopes in sequence space underpinned by binding-induced conformational switching.     

Smooth Functional Transition along a Mutational Pathway with an Abrupt Protein Fold Switch

Recent protein design experiments have demonstrated that proteins can migrate between folds through the accumulation of substitution mutations without visiting disordered or nonfunctional points in sequence space. To explore the biophysical mechanism underlying such transitions we use a three-letter continuous protein model with seven atoms per amino acid to provide realistic sequence-structure and sequence-function mappings through explicit simulation of the folding and interaction of model sequences. We start from two 16-amino-acid sequences folding into an α-helix and a β-hairpin, respectively, each of which has a preferred binding partner with 35 amino acids. We identify a mutational pathway between the two folds, which features a sharp fold switch. By contrast, we find that the transition in function is smooth. Moreover, the switch in preferred binding partner does not coincide with the fold switch. Discovery of new folds in evolution might therefore be facilitated by following fitness slopes in sequence space underpinned by binding-induced conformational switching.     

针对泛素化与去泛素化酶的化学探针

蛋白质泛素化对真核细胞内几乎所有生理过程都具备调控作用。新的泛素化与去泛素化酶的发现、功能机制研究及相关化学分子干预是该领域的重要科学命题。针对泛素化与去泛素化酶的化学探针开发促进了人们对于蛋白质泛素化的形成、募集、信号传导及脱除过程中所涉及生化事件的理解。本文总结了近年来针对泛素化与去泛素化酶化学探针的发展，归纳了不同类型探针的化学结构及合成方法，并讨论了它们的各类应用，包括筛查泛素依赖的信号传导系统、监控泛素相关酶活性、辅助泛素相关的识别和催化过程的分子机制解析等。     

Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

Identification of Functional Regions Defining Different Activity in Caspase-3 and Caspase-7 within Cells

Caspases are central to apoptosis, and the principal executioner caspases, caspase-3 and -7, were reported to be similar in activity, primary structure, and three-dimensional structure. Here, we identified different activity in caspase-3 and -7 within cells and examined the relationship between their structure and function using human cells expressing almost equal amounts of exogenous caspase-3, caspase-7, and/or chimeric constructs after down-regulation of endogenous caspase-3 and -7 expression. Caspase-3 (produced in human cells) showed much stronger cleaving activity than caspase-7 against a low molecular weight substrate in vitro dependent on four specific amino acid regions. Within cells, however, an additional three regions were required for caspase-3 to exert much stronger protease activity than caspase-7 against cellular substrates. Three of the former four regions and the latter three regions were shown to form two different three-dimensional structures that were located at the interface of the homodimer of procaspase-7 on opposite sides. In addition, procaspase-3 and -7 revealed specific homodimer-forming activity within cells dependent on five amino acid regions, which were included in the regions critical to the cleaving activity within cells. Thus, human caspase-3 and -7 exhibit differences in protease activity, specific homodimer-forming activity, and three-dimensional structural features, all of which are closely interrelated.     

Caspase-7: A protease involved in apoptosis and inflammation

Caspase-7 was considered to be redundant with caspase-3 because these related cysteine proteases share an optimal peptide recognition sequence and have several endogenous protein substrates in common. In addition, both caspases are proteolytically activated by the initiator caspase-8 and -9 during death receptor- and DNA-damage-induced apoptosis, respectively. However, a growing body of biochemical and physiological data indicate that caspase-7 also differs in significant ways from caspase-3. For instance, several substrates are specifically cleaved by caspase-7, but not caspase-3. Moreover, caspase-7 activation requires caspase-1 inflammasomes under inflammatory conditions, while caspase-3 processing proceeds independently of caspase-1. Finally, caspase-7 deficient mice are resistant to endotoxemia, whereas caspase-3 knockout mice are susceptible. These findings suggest that specifically interfering with caspase-7 activation may hold therapeutic value for the treatment of cancer and inflammatory ailments.     

Chemical,Target, and Bioactive Properties of Allosteric Modulation

Allosteric modulators are ligands for proteins that exert their effects via a different binding site than the natural (orthosteric) ligand site and hence form a conceptually distinct class of ligands for a target of interest. Here, the physicochemical and structural features of a large set of allosteric and non-allosteric ligands from the ChEMBL database of bioactive molecules are analyzed. In general allosteric modulators are relatively smaller, more lipophilic and more rigid compounds, though large differences exist between different targets and target classes. Furthermore, there are differences in the distribution of targets that bind these allosteric modulators. Allosteric modulators are over-represented in membrane receptors, ligand-gated ion channels and nuclear receptor targets, but are underrepresented in enzymes (primarily proteases and kinases). Moreover, allosteric modulators tend to bind to their targets with a slightly lower potency (5.96 log units versus 6.66 log units, p<0.01). However, this lower absolute affinity is compensated by their lower molecular weight and more lipophilic nature, leading to similar binding efficiency and surface efficiency indices. Subsequently a series of classifier models are trained, initially target class independent models followed by finer-grained target (architecture/functional class) based models using the target hierarchy of the ChEMBL database. Applications of these insights include the selection of likely allosteric modulators from existing compound collections, the design of novel chemical libraries biased towards allosteric regulators and the selection of targets potentially likely to yield allosteric modulators on screening. All data sets used in the paper are available for download.     

Biotin-Labeled Synthetic Oligodeoxyribonucleotides: Chemical Synthesis and Uses as Hybridization Probes

Abstract

Several biotinylated synthetic oligodeoxyribonucleotides have been selectively prepared by a simple and efficient chemical method. This procedure allows the specific and covalent coupling of one biotinmoiety to any 5′-kinased unprotected oliqodeoxyribonucleotide through an aminoalkylphosphoramide linker arm. The reactions are performed in aqueous solutions on unprotected oligonucleotides and proceed cleanly with good yields. This method is insensitive of the length of the polynucleotide, of the nucleotide sequence and of the nature of the 5′-terminal nucleotide.     

The Interactions of Enzyme and Chemical Probes with Inverted Repeats in Supercoiled DNA

Abstract

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as SI, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endo- nucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Mapping Flexibility and the Assembly Switch of Cell Division Protein FtsZ by Computational and Mutational Approaches

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal β-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.     

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr(178), generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. K(m) values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μm, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.     

Caspase-7 gene disruption reveals an involvement of the enzyme during the early stages of apoptosis

Caspases play a key role during apoptotic execution. In an attempt to elucidate the specific role of caspase-7 we generated a chicken DT40 cell line in which both alleles of the gene were disrupted. Viability assays showed that caspase-7-/- clones are more resistant to the common apoptosis-inducing drugs etoposide and staurosporine. Caspase-7-/- cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates poly(ADP-ribose) polymerase 1 and lamins B1 and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation compared with wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes.     

日本三角涡虫Caspase-7基因的克隆及功能研究

本文以日本三角涡虫Dugesia japonica为研究对象,通过c DNA末端快速扩增技术首次克隆得到了涡虫Caspase-7基因,全长935 bp,编码251个氨基酸,5'-UTR和3'-UTR长度分别为86 bp和93 bp。涡虫Caspase-7具有Caspase家族保守而关键的LSHG与QAC(Q/R) G两大结构域。此外,基于SWISS-MODEL同源建模对涡虫Caspase-7蛋白三维结构的预测和分析,发现其包含2个催化单位,且潜在催化位点由4个表面环构成。进化分析也显示,涡虫Caspase-7基因未与扁形动物门Platyhelminthes物种聚为一支,而与刺胞动物门Cnidaria水螅Hydra vulgaris有较近的亲缘关系,说明Caspase-7基因可能发生了趋同进化。本文通过实时荧光定量PCR检测了Caspase-7基因在涡虫再生不同时间段的表达变化,发现在涡虫切后再生6 h和3 d,Caspase-7基因表达量升高,这2个时间点是涡虫切后凋亡发生的2个高峰期。此外,通过喂食ds RNA干扰涡虫体内Caspase-7基因的表达,结果发现,干扰Caspase-7基因并不影响涡虫的再生,但再生完成后或未切割的成体涡虫在饥饿环境下,受Caspase-7基因干扰,虫体逐渐溶解直至死亡,而Djclg-3及PCNA基因的表达量也显著降低,说明干扰Caspase-7基因表达可能引起涡虫细胞凋亡与增殖减少,涡虫组织重塑失衡,导致虫体溶解并死亡。该结果揭示Caspase-7基因可能在涡虫组织重塑中起关键作用。     

Mutational and evolutionary analysis of an archaebacterial gene

The bacteriorhodopsin precursor gene of H. halobium is shown to contain 71 potential sites of mutation to premature stop codons, encompassing all detectable transitions and transversions. This same gene contains 24 potential frameshift hot-spots, all of the G and/or C type, nine possible dam methylation sites, and a unique mec methylation site which may act as a deamination mutagenesis hot-spot. While the gene contains pre-dominantly GC-rich codons, there is appreciable usage of other types of codons for particular amino acids. The bacteriorhodopsin precursor gene does not contain an abnormally high proportion of RNY-type, primitive codons, suggesting that even if distinct from the eubacteria, and an ancient group, modern-day archaebacteria have, nevertheless, undergone extensive evolution.