Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

A Non-canonical DNA Structure Enables Homologous Recombination in Various Genetic Systems

Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.Homologous recombination (HR)² is essential for gametogenesis during meiosis and plays an important role in the generation of genetic diversity, a process that is critical for natural selection. A general HR intermediate is the heteroduplex joint, which is formed between a single-stranded (ss) DNA tail derived from a double-stranded break and a homologous double-stranded (ds) DNA by homologous pairing (HP) (1, 2) and subsequent strand exchange (3, 4). HP is a switch from parental dsDNA base pairs to recombinant base pairs involving the ssDNA and the complementary strand of the dsDNA, which form the core of the recombination intermediate, and strand exchange is the unidirectional replacement of a dsDNA strand by the incoming ssDNA. The RecA/Rad51 family of proteins, which include bacterial RecA, archaeal RadA/Rad51, eukaryotic Rad51, and meiosis-specific Dmc1, are essential for HR in their respective organisms, and these proteins can promote ATP-dependent HP and ATP hydrolysis-dependent strand exchange in vitro (see Refs. 5–10, for reviews). In HP, ATP-bound RecA first binds to ssDNA, and this ssDNA·RecA complex then interacts with dsDNA without homologous recognition. Within the RecA·ssDNA·dsDNA complex, a homologous region is identified (11). The base pair switch in HP is formally carried out by base rotation or base flipping (rotation around the base-sugar bond), either of which requires the expansion of the spaces between neighboring bases or base pairs (see Refs. 7 and 12).Electron microscopic studies have shown that RecA/Rad51 proteins form a well conserved right-handed helical filament around ssDNA or dsDNA (13–15). In the absence of ATP, these proteins assemble as a shorter inactive filament (helical pitch, ≈65–85 Å) (16, 17). In the presence of ATP (or ATPγS, a non-hydrolyzable ATP analogue), the filament adopts an extended active conformation with a helical pitch of ≈95 Å, and the contour length of ssDNA and dsDNA within the active filament is elongated to the same extent (13–15, 17, 18). This equalized elongation has been inferred to widen the spacing between bases of ssDNA and dsDNA equally in the nucleoprotein filament to facilitate the homologous alignment of both DNA substrates to achieve base pair switching (13). Previously, we analyzed the three-dimensional structure of the RecA·ssDNA complex in the presence of ATPγS by NMR, which showed that the axial rise per ssDNA base was extended to nearly 5 Å (19), and that the interconversion of sugar puckers induced horizontal base rotation (20). On the basis of these results, and as there was no other structural information at that time, we proposed a base rotation mechanism to explain the base pair switch in HP by assuming that the ssDNA and dsDNA were extended equally and uniformly (20). Recently, the crystal structure of the RecA·ssDNA complex has revealed a non-uniformly extended structure for the ssDNA (21). The crystal structure contains “a three-nucleotide segment” (triplet) region and “a long untwisted inter-nucleotide” (inter-triplet) region (see Fig. 4). However, it remains unclear which structure contributes to HP and how it does so.Open in a separate windowFIGURE 4.Comparison of the solution and crystal structures of ssDNA bound to RecA. A, superimposition of the solution structure of RecA-d(TACG) (19) (in magenta) and four DNA residues in the RecA₅-(dT)₁₅ crystal structure (21) (in cyan). The crystal structure is similar to the solution structure. D1, D2, and D3 indicate distances between adjacent bases (see also 18, 20). Actually, the dsDNA was shown to be untwisted (unwound) within the homology-independent RecA-ssDNA-dsDNA intermediate of HP described above (22, 23). HP mediated by RecA or Rad51 was shown to be extensively stimulated by negative supercoiling of the dsDNA substrate in vitro (24, 25). This is probably because negative supercoils in the dsDNA substrate would compensate for the positive supercoils generated by the untwisting for HP. Closed circular dsDNA isolated from living cells, including DNA from bacteria, nuclei, or mitochondria of eukaryotic cells, is similarly supercoiled. However, in vivo, the supercoils of cellular dsDNA are relaxed by nucleosome assembly in eukaryotic nuclei and by the binding of HU (26) and/or other DNA-binding proteins in bacteria. Thus, dsDNA in vivo is an unfavorable substrate for HP mediated by RecA/Rad51 family proteins.In mitochondria, which do not have RecA/Rad51 family proteins, negative supercoils are relaxed by the binding of TFAM (in mammals) or Abf2 (in yeast) (27, 28). In this in vivo dsDNA state, Mhr1, an ATP-independent HP protein required for mitochondrial HR in the budding yeast Saccharomyces cerevisiae (29) catalyzes HP without the net untwisting of dsDNA, i.e. Mhr1 catalyzes HP with relaxed closed circular dsDNA with similar efficiency as with dsDNA lacking topological constraints (linear dsDNA and closed circular dsDNA in the presence of a topoisomerase) (30). Furthermore, in contrast to what is observed for RecA/Rad51, Mhr1-catalyzed HP is prevented by negative dsDNA supercoiling. The absence of net untwisting of dsDNA appears at first glance to mean HP without the extension of the parental dsDNA. However, HP requires the expansion of inter-base pair spaces for base pair switching as described above, and thus we proposed that right-handed wrapping of dsDNA around Mhr1 with an extended and untwisted configuration allows base rotation for HP (30). However, it remained to be experimentally determined whether Mhr1 and RecA/Rad51 share a common or different mechanism for HP.In addition to Mhr1, several proteins that promote HP in vitro in the absence of nucleotide cofactors have been identified. These include the human (hs) Xrcc3·Rad51c/Rad51L2 complex (human Rad51 paralogues; 31), hsRad52 (32), Escherichia coli (ec) phage λ β-protein (33), ecRecT (a homologue of λ β-protein 34), ecRecO (35), and Ustilago maydis Brh2 (36). Some of these proteins are termed recombination mediators, but we refer to them as ATP-independent HP proteins for the purpose of this study (supplemental Fig. S1). In contrast to RecA/Rad51 family members, ATP-independent HP proteins, except for those in the Xrcc3·Rad51C complex, do not exhibit any amino acid sequence homology with RecA/Rad51 proteins or other ATP-independent HP proteins. In addition, ATP-independent HP proteins exhibit significantly different quaternary structures (31, 32, 36–40). The N-terminal domain of hsRad52 forms an undecameric ring around which ssDNA and/or dsDNA wrap(s) (32, 41), and the interaction of closed circular dsDNA with hsRad52 generates negative supercoils (41), whereas binding to RecA generates positive supercoils in this substrate. On the other hand, like RecA, RecT was shown to untwist dsDNA during HP (42). The binding of dsDNA to Mhr1 causes neither untwisting nor twisting (30). Thus, the properties of HP proteins vary considerably except for their HP activities.In this study, we questioned whether the extended structure of ssDNA as seen in the RecA·ssDNA complex is conserved among HP proteins, or whether each HP protein uses a different principle to promote HP. If the extended structure is a common determinant of HP, the different HP proteins are likely to use a common mechanism to promote HP, but their variation may reflect requirements for optimizing HP in different cellular environments. Thus, we focused on the structure of the HP protein-bound ssDNA, an HP intermediate. We determined the three-dimensional structures of ssDNA bound to Mhr1 and of three other evolutionarily distinct HP proteins, ecRecT (ATP-independent, from the λ-like cryptic prophage Rac of E. coli, involved in plasmid HR (43)), Thermus thermophilus (tt) RecO (ATP-independent HP protein in bacteria), and hsRad51 (ATP-dependent, human nuclear homologue of RecA) and compared them with ssDNA bound to ecRecA (E. coli, the prototype of the RecA family; supplemental Fig. S1). This is the first demonstration that diverse HP proteins, both ATP-dependent (RecA/Rad51) and ATP-independent (Mhr1, RecO, RecT), use the non-canonical extended DNA structure as a common intermediate for HP, and this suggests that they use a common mechanism for HP.