Decreased Protein Stability Research Articles

Identification of a new isoform of the murine Sh2d1a gene and its functional implications

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is a Src homology (SH) domain 2-containing intracellular adaptor protein that is predominantly expressed in the hematopoietic system by T lymphocytes and NK cells. SAP protein is encoded by the SH2D1A gene located on the X chromosome. Loss-of-function mutations in SAP cause the X-linked lymphoproliferative disease (XLP), a severe immunodeficiency characterized by heightened susceptibility to Epstein-Barr virus and impaired humoral immunity. Normal individuals express several functional and non-functional isoforms of SAP as a result of alternative splicing. In this study, we identify a cryptic exon in the murine Sh2d1a gene. At the mRNA level, the new isoform of SAP (SAP-2) that includes this new exon is widely expressed in lymphoid tissues by C57BL/6 and 129 strains of inbred mice. SAP-2 accounts for approximately 1%-3% of total SAP transcripts, and it is dynamically regulated during lymphocyte activation. At the protein level, the SAP-2 isoform is a 144 amino-acid protein. Compared to the dominant 126 aminoacid SAP-1 isoform, the additional 18 amino acids are inserted into a structural region that is critical for phosphotyrosine binding. Our functional analysis in vitro indicates that SAP-2 is a non-functional isoform due to decreased protein stability. Thus, both human and mouse have multiple SAP splice isoforms that may or may not function. Modulation of relative proportions of these isoforms is potentially a mechanism whereby cells can regulate SAP-mediated biological activities.

Abstract P3-03-01: The role of a newly discovered N-terminal methyltransferase in breast cancer

Abstract The recently discovered N-terminal methyltransferase, NRMT, is the first eukaryotic N-terminal methyltransferase to be identified. NRMT is a highly conserved, ubiquitously expressed, nuclear MT. After methionine cleavage, it methylates the X-P-K amino-terminal consensus sequence of its substrates. Loss of N-terminal methylation has been associated with decreased ability to bind to DNA and decreased protein stability. Many NRMT targets are known tumor suppressors or oncogenes, including DDB2, RB, SET, and NEMO. Knockdown of NRMT in HEK cells results in increased cell proliferation and multi-spindle formation, which are characteristics of malignant cells. In addition, microarray analysis has shown that NRMT expression can be either up or down regulated in a variety of human cancers. Our preliminary data in human breast cancer samples verifies the microarray data and shows that NRMT mRNA levels are both significantly increased or decreased in a patient specific manner. NRMT expression levels do not seem dependent on the type of tumor, therefore, we hypothesize that NRMT expression levels may be dependent on the endocrine sensitivity of the tumor. To test if NRMT activity differentially affects endocrine sensitive and endocrine resistant cell types in vitro, we knocked down NRMT expression in a panel of human breast cancer cell lines with decreasing endocrine sensitivity using shRNA. Cells expressing the shRNA against human NRMT (or mouse NRMT as a control) were then assayed for proliferation and migration potential. Knockdown of NRMT significantly increased proliferation of the endocrine sensitive MCF-7 and T47D cancer cell lines over a five-day time course. In contrast, NRMT knockdown only slightly inhibited LCC9 (an antiestrogen-resistant MCF-7 variant line) proliferation and had no effect on endocrine resistant MDA-MB-231 cells. NRMT knockdown also significantly inhibited MCF-7 cell migration at 72 hours but only slightly affected LCC9 cells and had no affect on the MDA-MD-231 cells. These data indicate that NRMT loss early in tumorigenesis has the potential to increase the growth rate of endocrine sensitive primary tumors but has little affect on endocrine resistant cells. We are now interested in determining if we can increase endocrine resistant cell proliferation and migration through NRMT overexpression, thus explaining the microarray profiles. Taken together these studies indicate that N-terminal methylation by NRMT is a biologically important post-translational modification with regard to cell division and tumorigenesis in breast cancer. Next, we will test if NRMT loss can confer endocrine resistance to endocrine sensitive cells and use a murine mammary gland transplantation and regeneration model to study the effect of NRMT knockdown on mammary tumorigenesis and metastasis in vivo. Understanding the general biological function of N-terminal methylation and its relationship to endocrine sensitivity could lead to the use of NRMT expression levels as a biomarker for tumors likely to develop resistance to endocrine therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-01.

Role of a Conserved Residue R780 in Escherichia coli Multidrug Transporter AcrB

Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.

Porphyromonas gingivalis lipopolysaccharide increases lipid accumulation by affecting CD36 and ATP-binding cassette transporter A1 in macrophages

Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) promotes macrophage-derived foam cell formation, however, the mechanisms are not well established. In macrophages, lipid uptake is mediated by scavenger receptors including SR-A and CD36, while the cholesterol efflux is mediated by ATP-binding cassette transporter G1 (ABCG1), ABCA1 and SR-BI. We further investigated the mechanisms underlying the dysregulation by P. gingivalis LPS of these regulators resulting in the promotion of lipid accumulation in THP-1-derived macrophages. Our results showed that P. gingivalis LPS exacerbated lipid accumulation in oxidized low-density lipoprotein (oxLDL)-treated macrophages. However, cholesterol efflux was inhibited by P. gingivalis LPS in THP-1-derived macrophages. In oxLDL-untreated macrophages, P. gingivalis LPS treatment caused an increase in CD36 mRNA and protein levels, and a decrease in ABCA1 mRNA and protein levels, while having no effect on SR-A, SR-BI or ABCG1 expression. Upregulation of CD36 by P. gingivalis LPS resulted from activation of c-Jun/AP-1, and this was confirmed by the inhibition of increased CD36 expression after AP-1 inhibition using SP600125. However, the decreased protein stability of ABCA1 by P. gingivalis LPS was a result of increased calpain activity. Moreover, small hairpin RNA (shRNA) targeting heme oxygenase-1 (HO-1) augmented the P. gingivalis LPS-induced atherogenic effects on the expression of c-Jun/AP-1, CD36, ABCA1 and calpain activity. Accordingly, P. gingivalis LPS-regulated promotion of lipid accumulation in foam cells was also exacerbated by HO-1 shRNA. These results indicate that P. gingivalis LPS confers a exacerbation effect on the formation of foam cells by a novel HO-1-dependent mediation of cholesterol efflux and lipid accumulation in macrophages.

Ligand binding and aggregation of pathogenic SOD1.

Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer–dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein–ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II–strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

A Conserved Structural Determinant Located at the Interdomain Region of Mammalian IRE1α

The UPR sensor inositol‐requiring enzyme 1α (IRE1α) is a bifunctional enzyme containing both kinase and RNase domains that are important for trans‐autophosphorylation and Xbp1 mRNA splicing, respectively, in response to ER stress. However, the amino acid residues important for structural integrity remain largely unknown. Here, through analysis of IRE1α mutants associated with human somatic cancers, we have identified a highly conserved proline residue at position 830 (P830) that is critical for its structural integrity, hence the activation of both kinase and RNase domains. Structural analysis revealed that P830 may form a highly conserved structural linker with adjacent tryptophan and tyrosine residues at positions 833 and 945 (W833 and Y945), thereby bridging the kinase and RNase domains. Indeed, mutation of P830 to leucine (P830L) completely abolished the kinase and RNase activities, significantly decreased protein stability and prevented oligomerization of IRE1α upon ER stress; similar observations were made for mutations of W833 to alanine (W833A) and to a lesser extent for Y945A. Our finding may facilitate the identification of small molecules to specifically compromise IRE1α function.

Identification of FoxM1 As Therapeutic Target in TKI-Resistant Ph+ ALL

Abstract 874 Background:Despite initial responsiveness of primary Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) to tyrosine kinase inhibition (TKI), the majority of patients will relapse and develop TKI-resistant disease. Foxm1 belongs to the forkhead box transcription factor family and is a key regulator of malignant growth by promoting cell cycle and survival through increased DNA damage repair. Foxm1 has been implicated in the progression and chemoresistance in a wide range of solid tumors, including hepatocellular carcinoma and breast cancer. Foxm1 is expressed in dividing cells and regulates the expression of critical regulators for G2/M entry of the cell cycle including Cdc25B, cyclin-B1, Plk-1 and Aurora B kinase. In addition it decreases protein stability of p27kip and p21cip and regulates the expression of antioxidant defense machinery of the cell, e.g. by superoxide dismutase expression. Results:We compared Foxm1 expression levels in Ph+ ALL patient samples and CD19+ B cell precursors from healthy donors and found 12-fold higher levels in the leukemic cells (p=0.011). More importantly, Foxm1 levels at the time of diagnosis in a clinical trial for patients with high risk acute lymphoblastic leukemia (ALL) were predictive of poor outcome (COG P9906; n=207). Comparative analysis of microarray data from matched sample pairs at diagnosis and relapse revealed a significant upregulation of Foxm1 in the relapse samples (n=42; p=0.0025). To further study the role of Foxm1 in Ph+ ALL, we developed a genetic model for inducible inactivation of Foxm1 in Ph+ ALL. To this end, B cell precursors of Foxm1fl/fl mice were transformed with BCR-ABL1 and transduced with a tamoxifen (4-OHT)-inducible Cre. Interestingly, 4-OHT-mediated deletion of Foxm1 resulted in reduced cell viability and an arrest in G0/G1 with a significant decrease of the S-phase of the cell cycle following deletion of Foxm1. The ability to form colonies in vitro was significantly decreased by deletion of Foxm1. In addition, Foxm1−/− ALL cells revealed a strikingly higher sensitivity towards TKI-treatment (Imatinib dose-response curve) compared to the control cells. As a potential therapeutic agent to pharmacologically inhibit Foxm1 function, we evaluated the effects of a previously described ARF peptide that binds and inhibits Foxm1 function. We treated TKI-resistant (BCR-ABL1T315I) and TKI-sensitive patient-derived xenograft Ph+ ALL cells with various ARF peptide concentrations and found significant growth inhibition after 72h (IC50 16.8±4.3μM, n=4), regardless of TKI responsiveness. In addition, treatment of ARF peptide in combination with TKI reduced the viability from 65.7%±1.7 after TKI treatment alone (10μM) to 19%±0.8 after 48h (TKI 10μM ARF peptide 12μM). Ph+ ALL cells treated with similar concentrations of a mutated ARF control peptide revealed 77%±0.9 viable cells and ARF peptide treatment alone decreased the viability to 29.6%±0.4. Hence treatment with the ARF peptide alone induces apoptosis in patient-derived Ph+ ALL cells and enhances the effect of TKI, which confirms the findings of the ALL mouse model for human Ph+ ALL xenografts. In a complementary approach, we used the natural antibiotic Thiostrepton, which functions via Foxm1 blockade. To test the ability of Thiostrepton as a potential anti-leukemia agent, we studied patient-derived TKI-resistant (BCR-ABL1T315I) Ph+ ALL cells. Treatment of these patient derived Ph+ALL cells induced cytotoxicity in nanomolar concentrations of Thiostrepton along with a significant downregulation of Foxm1 protein levels. By contrast, Non-BCR-ABL1 tumor cells including lymphoma cells were not responsive to Thiostrepton treatment at similar concentrations. Conclusion:Our analyses reveal that Foxm1 is a valid therapeutic target for the treatment of TKI sensitive and resistant Ph+ ALL, including BCR-ABL1T315I. We show that Foxm1 has a crucial function in Ph+ ALL and impacts a) leukemia proliferation, b) colony formation, and c) TKI-resistance. These findings identify Foxm1 a rational target for combination therapy with TKI or as a single agent for TKI-resistant Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.

Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.

Dystrophin’s Tandem Calponin-Homology Domains: Is the Case Closed?

Dystrophin’s Tandem Calponin-Homology Domains: Is the Case Closed?

Abstract 63: Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Cardiomyopathy Affects Protein Structure, Decreases Protein Stability and Increases Protein Unfolding

A genetic mutation in dystrophin that replaces lysine at amino acid position 18 by an asparagine (K18N) results in X-linked cardiomyopathy (XCM), however, the underlying molecular mechanisms are not known. Such knowledge might help in developing effective therapies to treat XCM. The first step in understanding disease-triggering mechanisms is to probe how mutation affects protein structure and function. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD) of dystrophin. Here, we examined the effect of K18N on the structure, stability, and function of N-ABD. Fig. 1A shows the 15 N- 1 H HSQC NMR spectrum before and after the mutation. Each crosspeak in the spectrum represents the structure around an amino acid. Mutation affects the position of nearly all the crosspeaks. These changes are not just located at the mutation site, but were observed in regions far away from the mutation site and also in the three actin binding regions with which N-ABD interacts with actin. In addition, the mutation makes the protein more dynamic. These structural changes result in decreased actin binding function of N-ABD. Also, the K18N mutation decreases the stability by ΔΔG = 5.3 kcal/mol (Fig. 1B) and increases the rate of unfolding by 3.2 times (Fig. 1C), which exponentially increase the relative population of unfolded state that is more prone to proteolysis and hence will result in decreased levels of the functional protein. These results indicate that the physical mechanism by which the K18N mutation triggers the disease might be by affecting protein structure leading to decreased actin binding and by decreasing the net protein levels leading to decreased net function.

Relationship between enzyme properties and disease progression in Canavan disease

Canavan disease (CD) is a fatal neurological disorder caused by defects in the gene that encodes for a critical metabolic enzyme. The enzyme aspartoacylase catalyzes the deacetylation of N-acetylaspartate to produce acetate required for fatty acid biosynthesis in the brain. The loss of aspartoacylase activity leads to the demyelination and disrupted brain development that is found in CD patients. Sixteen different clinical mutants of aspartoacylase have been cloned, expressed and purified to examine their properties and the relationship between enzyme properties and disease phenotype. In contrast to numerous cell culture studies that reported virtually complete loss of function, each of these purified mutant enzymes was found to have measureable catalytic activity. However, the activities of these mutants are diminished, by as little as three-fold to greater than 100-fold when compared to the native enzyme. Many of these mutated enzyme forms show decreased thermal stability and an increased propensity for denaturation upon exposure to urea, but only four of the 16 mutants examined showed both diminished thermal and diminished conformational stability. Significantly, each of these lower stability mutants are responsible for the more severe phenotypes of CD, while patients with milder forms of CD have aspartoacylase mutants with generally high catalytic activity and with either good thermal or good conformational stability. These results suggest that the loss of catalytic function and the accumulation of N-acetylaspartate in Canavan disease is at least partially a consequence of the decreased protein stability caused by these mutations.

Sampling the Conformational Energy Landscape of a Hyperthermophilic Protein by Engineering Key Substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.

Role of the N- and C-Terminal Helices in Apolipophorin III Stability and Lipid Binding

Apolipophorin III (apoLp-III) from the insect Locusta migratoria is a model apolipoprotein to study their structure-function relationship. In contrast to the common 4-helix bundle motif, apoLp-III is a bundle of five amphipathic α-helices. We hypothesized that the 5-helix bundle of apoLp-III may have evolved to modulate lipid-binding activity. To verify this, N- and C-terminal helix deletion mutants were designed. The N-terminal deletion mutant was expressed and purified from E.coli. However, bacteria did not produce the C-terminal helix deletion mutant. Therefore, valine 131, which resides in the loop connecting helix-4 and −5, was replaced by methionine. Cleavage by CNBr then produced the C-terminal deletion variant. in addition, five truncation mutants in the C-terminal α-helix were engineered by introducing stop codons at Q136, E140, E144, Q150 and Q154, to create shortened versions of the protein. The mutant proteins truncated at E144, Q150 and Q154 were successfully expressed and purified but no protein was produced when apoLp-III was truncated at Q136 or E140. Guanidine denaturation analysis showed a decreased stability for the N- and C-terminal helix deletion mutants as indicated by the midpoint of denaturation (0.19 and 0.23 M for the deletion mutants and 0.50 M for wild-type apoLp-III). Truncation at E144, Q150 and Q154 also resulted in a decrease in protein stability with midpoints of 0.23, 0.35, and 0.36 M, respectively. Far UV circular dichroism showed a significant reduction in α-helical content for all mutants. Lipid binding analysis using dimyristoylphosphatidylcholine vesicles showed that the mutant proteins formed discoidal protein-lipid complexes at much higher rates compared to wild-type apoLp-III. The results show that deletion of a terminal α-helix result in an increased lipid binding but this comes at the cost of a decrease in protein stability.

A Conserved Structural Determinant Located at the Interdomain Region of Mammalian Inositol-requiring Enzyme 1α

Inositol-requiring enzyme 1α (IRE1α), an endoplasmic reticulum-resident sensor for mammalian unfolded protein response, is a bifunctional enzyme containing kinase and RNase domains critical for trans-autophosphorylation and Xbp1 mRNA splicing, respectively, in response to endoplasmic reticulum stress. However, the amino acid residues important for its function and activation remain largely unexplored. Here, through analysis of IRE1α mutants associated with human somatic cancers, we have identified a highly conserved proline residue at position 830 (Pro(830)) that is critical for its structural integrity and hence, the activation of both kinase and RNase domains. Structural analysis revealed that Pro(830) may form a highly conserved structural linker with adjacent tryptophan and tyrosine residues at positions 833 and 945 (Trp(833) and Tyr(945)), thereby bridging the kinase and RNase domains. Indeed, mutation of Pro(830) to leucine (P830L) completely abolished the kinase and RNase activities, significantly decreased protein stability, and prevented oligomerization of IRE1α upon ER stress; similar observations were made for mutations of Trp(833) to alanine (W833A) and to a lesser extent for Y945A. Our finding may facilitate the identification of small molecules to compromise IRE1α function specifically.

The Congenital Cataract-Linked G61C Mutation Destabilizes γD-Crystallin and Promotes Non-Native Aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

Nonconserved Lysine Residues Attenuate the Biological Function of the Low-Risk Human Papillomavirus E7 Protein

The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures.

Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima

The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D: -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.

The Effect of Protein Stability on Interactions of Apomyoglobin Forms with Phospholipid Membranes

The protein structure can be strongly influenced by phospholipid membranes. As it follows from our papers, apomyoglobin structure undergoes a transition from the native to some intermediate state upon interaction with small negatively charged phospholipid vesicles acting as a moderately denaturing agent. In this work, interaction of apomyoglobin and its mutant forms with artificial membranes is studied by tryptophan fluorescence and CD in the far UV-region. It is shown that a negatively charged phospholipid membrane can structure the unfolded protein into the same intermediate state. The nature of this phenomenon consists in selective stabilization of the intermediate state structure. The rate of interactions between apomyoglobin mutant forms and phospholipid membranes depends mainly on the protein molecule stability, as well as on the charge of the membrane surface and the phospholipid vesicles concentration. This rate increases with decreasing protein stability. The importance of the obtained results for the folding of membrane proteins and the choice of the pathway for target delivery of protein drugs are discussed. This work was supported partly by the Howard Hughes Medical Institute Award 55005607 to A.V. Finkelstein, by the RAS Program “Molecular and Cellular Biology”, by Federal Agency for Science and Innovations 02.740.11.0295, and Program of Scientific Schools 2791.2008.4.

Packing of the Extracellular Domain Hydrophobic Core Has Evolved to Facilitate Pentameric Ligand-gated Ion Channel Function

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain β-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA(A) receptors show that increasing the β-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic β-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.

Missense Mutations in N-Terminal Actin Binding Domain of Dystrophin that Trigger Muscular Dystrophy Decrease Protein Stability and Lead to Cross-β Aggregates

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations - L54R, A168D, A171P, and Y231N - on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR 15N-1H HSQC fingerprint region, and its rotational correlation time calculated from NMR longitudinal (T1) and transverse (T2) relaxation experiments. Compared to WT, three mutants - L54R, A168D, and A171P - show a decreased α-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a ‘molten globule’ state. In contrast, Y231N has an α-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and congo red absorption spectral shift and birefringence show that these aggregates contain intermolecular cross-β structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.