How DNA Polymerase X Preferentially Accommodates Incoming dATP Opposite 8-Oxoguanine on the Template

The modified base 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoG_syn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoG_syn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoG_syn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:A_syn) to extreme (i.e., oxoG_anti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function.     

A de novo protein binding pair by computational design and directed evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

The promyelocytic leukemia protein protects p53 from Mdm2-mediated inhibition and degradation

The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.     

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase beta. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.     

Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle

Certain PKC isoforms are stimulated by insulin and interact with IR as well as with IRS, but it is still not clear if specific PKC isoforms regulate IR signaling directly or through IRS-1. PKCalpha may regulate IRS activity in response to insulin. We investigated the possibility that PKCalpha may be important in insulin signaling. Studies were conducted on skeletal muscle in adult mice and on L6 skeletal cells. PKCalpha is constitutively associated with IRS-1, and insulin stimulation of PKCalpha causes disassociation of the two proteins within 5 min. Blockade of PKCalpha inhibited insulin-induced disassociation of PKCalpha from IRS1. Selective inhibition of PKCalpha increased the ability of insulin to reduce blood glucose levels. Insulin stimulation activates PKB and increases the association of PKCalpha with PKB. Blockade of PKCalpha increased threonine phosphorylation of PKB. We suggest that PKCalpha regulates insulin signaling in skeletal muscle through its disassociation from IRS-1 and association with PKB.     

The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.     

Tensile Mechanical Properties of Swine Cortical Mandibular Bone

Temporary orthodontic mini implants serve as anchorage devices in orthodontic treatments. Often, they are inserted in the jaw bones, between the roots of the teeth. The stability of the mini implants within the bone is one of the major factors affecting their success and, consequently, that of the orthodontic treatment. Bone mechanical properties are important for implant stability. The aim of this study was to determine the tensile properties of the alveolar and basal mandible bones in a swine model. The diametral compression test was employed to study the properties in two orthogonal directions: mesio-distal and occluso-gingival. Small cylindrical cortical bone specimens (2.6 mm diameter, 1.5 mm thickness) were obtained from 7 mandibles using a trephine drill. The sites included different locations (anterior and posterior) and aspects (buccal and lingual) for a total of 16 specimens from each mandible. The load-displacement curves were continuously monitored while loading half of the specimens in the oclluso-gingival direction and half in the mesio-distal direction. The stiffness was calculated from the linear portion of the curve. The mesio-distal direction was 31% stiffer than the occluso-gingival direction. The basal bone was 40% stiffer than the alveolar bone. The posterior zone was 46% stiffer than the anterior zone. The lingual aspect was stiffer than the buccal aspect. Although bone specimens do not behave as brittle materials, the diametral compression test can be adequately used for determining tensile behavior when only small bone specimens can be obtained. In conclusion, to obtain maximal orthodontic mini implant stability, the force components on the implants should be oriented mostly in the mesio-distal direction.