Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal subunit enzyme of human small intestinal maltase-glucoamylase (rhMGAM-N) was used to explore digestion of native starches from different botanical sources. The susceptibilities to enzyme hydrolysis varied among the starches. The rate and extent of hydrolysis of amylomaize-5 and amylomaize-7 into glucose were greater than for other starches. Such was not observed with fungal amyloglucosidase or pancreatic alpha-amylase. The degradation of native starch granules showed a surface furrowed pattern in random, radial, or tree-like arrangements that differed substantially from the erosion patterns of amyloglucosidase or alpha-amylase. The evidence of raw starch granule degradation with rhMGAM-N indicates that pancreatic alpha-amylase hydrolysis is not a requirement for native starch digestion in the human small intestine.     

Structural Analysis of the Interactions Between Hsp70 Chaperones and the Yeast DNA Replication Protein Orc4p

Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL₄) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p₂-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL₄ motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL₄ selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL₄ results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL₄ motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734 bp and encodes 578 amino acids with a molecular mass of 61,757 Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.     

Directed Evolution of the DnaK Chaperone: Mutations in the Lid Domain Result in Enhanced Chaperone Activity

We improved the DnaK molecular chaperone system for increased folding efficiency towards two target proteins, by using a multi-parameter screening procedure. First, we used a folding-deficient C-terminal truncated chloramphenicol acetyl transferase (CAT_Cd9) to obtain tunable selective pressure for enhanced DnaK chaperon function in vivo. Second, we screened selected clones in vitro for CAT_Cd9 activity after growth under selective pressure. We then analyzed how these variants performed as compared to wild type DnaK towards folding assistance of a second target protein; namely, chemically denatured firefly luciferase. A total of 11 single point DnaK mutants and 1 truncated variant were identified using CAT_Cd9 as the protein target, while 4 of the 12 selected variants showed improved luciferase refolding in vitro. This shows that improving the DnaK chaperone by using a certain target substrate protein, does not necessarily result in a loss or reduction in its ability to assist other proteins. Of the 12 identified mutations, half were clustered in the nucleotide binding domain, and half in the lid domain (LD) of DnaK. The truncated variant is characterized by a 35-residue C-terminal truncation (Cd35) and exhibited the highest improvement for luciferase refolding. Cd35 showed a 7-fold increase in initial refolding rate for denatured luciferase and resulted in a 5-fold increase in maximal luminescence as compared to wild type DnaK. Given that the best in vitro performing mutants contained LD substitutions, and that the LD is not involved in ATP binding, ATP hydrolysis or client protein association, but is involved in allosteric regulation of the chaperone cycle, we propose that improved DnaK variants result in changes to allosteric domain communication, ultimately retuning the ATP-dependent chaperone cycle.     

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.     

Molecular basis for the glycosphingolipid-binding specificity of α-synuclein: key role of tyrosine 39 in membrane insertion

Cell surface glycosphingolipids (GSLs) including gangliosides play a key role in the regulation of the conformation, oligomerization, and fibrillation of amyloidogenic proteins. Correspondingly, most amyloidogenic proteins possess a functional GSL-binding motif (GBM). Sequence alignments of GSL-binding proteins against the GBM of α-synuclein allowed the establishment of a consensus GBM sequence defined as K/H/R/-X(1-4)-Y/F-X(4-5)-K/H/R, where at least one of the X(1-4) residues is glycine. The GBMs of α-synuclein (34-KEGVLYVGSKTK-45) and Alzheimer's disease β-amyloid peptide (Aβ) (5-RHDSGYEVHHQK-16) consist of a structurally related loop centered on tyrosine (Y39 for α-synuclein, Y10 for Aβ). Surface pressure measurements of GSL monolayers at the air-water interface allowed us to determine the following order for α-synuclein-GSL interactions: GM3 > Gb3 > GalCer-NFA > GM1 > sulfatide > GalCer-HFA > LacCer > GM4 > GM2 > asialo-GM1 > GD3, indicating a marked preference for GSLs with one, three, or five sugar units. The insertion of α-synuclein into sphingomyelin-containing monolayers was strongly stimulated by the presence of GM3. This effect was not observed with phosphatidylcholine monolayers, suggesting that the ganglioside facilitated the insertion of α-synuclein into raft-like membrane domains. Molecular dynamics simulations suggested that the side chain of Y39 was deeply inserted between GM3 head groups. Monolayer experiments with mutant GBM peptides showed that Y39, K34, and K45 were important for GM3 binding, whereas only Y39 appeared critical for GM1 recognition. The interaction of Aβ 5-16 with GM1 involved R5, H13, H14, and K16, but not Y10. These data indicate that subtle amino acid variations in the consensus GBM of α-synuclein and Aβ conferred distinct GSL-binding properties.     

Allostery in Hsp70 Chaperones Is Transduced by Subdomain Rotations

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's.It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD?Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.     

Construction of combinatorial library of starch-binding domain of Rhizopus oryzae glucoamylase and screening of clones with enhanced activity by yeast display method

Most of the glucoamylases (GA), which catalyze the hydrolysis of -1,4 and -1,6 glycosidic linkages, have a distinct region called a starch-binding domain (SBD). We have developed a powerful method for screening a library of GA mutants by a combination of GA display and SBD mutagenesis on the yeast-cell surface. In the case of Rhizopus oryzae glucoamylase (RoGA), three amino acids (63S, 71T, 73S) of the SBD were combinatorially mutated to enhance the degradation activity toward cooked corn starch and the mutated RoGAs were displayed on yeast-cell surface by cell-surface engineering. After the first screening by halo assay using an iodine-starch reaction, about 200 of the 8000 colonies formed clear halos. Incubation of the yeast with the mutated and displayed RoGAs caused direct degradation of cooked corn starch. Repeated screening revealed that some of the mutants produced a degradation rate around 1.4-fold higher than did wild type. The results obtained from the DNA sequences of the mutated SBDs indicated that amino-acid residues with a carbonyl group (D, E, Q, N) in the SBD enhance the degradation ability of the GA by enhancing the binding activity of the SBD.     

NMR analysis of a kinetically trapped intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase

A thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase refolds into a kinetically trapped metastable intermediate when subjected to a rapid lowering of temperature. We attempted to characterise this intermediate using multidimensional NMR spectroscopy. The ¹H-¹⁵N heteronuclear single quantum coherence spectrum after a rapid temperature decrease (the spectrum of the intermediate) showed good chemical shift dispersion but was significantly different from that of the native state, suggesting that the intermediate adopts a nonnative but well-structured conformation. Large chemical shift changes for the backbone amide protons between the native and the intermediate states were observed for residues in the β-sheet consisting of strands 2, 3, 5, 6, and 7 as well as in the C-terminal region. These residues were found to be in close proximity to aromatic residues, suggesting that the chemical shift changes are mainly due to ring current shifts caused by the aromatic residues. The two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy experiments showed that the intermediate contained substantial, native-like NOE connectivities, although there were fewer cross peaks in the spectrum of the intermediate compared with that of the native state. It was also shown that there were native-like interresidue NOEs for residues buried in the protein, whereas many of the NOE cross peaks were lost for the residues involved in a surface-exposed aromatic cluster. These results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing.