Stretch Of Amino Acids Research Articles

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage. First, an unconventional “bipartite-like” nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full-length MCM9 for proper DNA repair.

Abstract PO-13: Structural requirements for GRK2-mediated inhibition of the MALT1 proto-oncoprotein

Abstract Stimulation of the B-cell or T-cell receptor triggers assembly of an intracellular complex composed of three proteins, CARMA1, BCL10, and MALT1 (CBM), which then induces activation of the NF-κB transcription factor and lymphocyte proliferation. Dysregulated CBM signaling, which occurs as a result of several gain-of-function mutations, underlies a variety of lymphoid malignancies including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL). Multiple studies established that inhibiting MALT1 reduces growth and viability of ABC-DLBCL. We recently identified G-protein-coupled receptor kinase 2 (GRK2), one of seven members of the GRK family, as a novel MALT1-binding protein and demonstrated that GRK2 inhibits MALT1-dependent NF-κB activation. Further, knockdown of GRK2 in ABC-DLBCL enhances tumor growth, suggesting that GRK2 may act as a tumor suppressor by binding and inhibiting MALT1. As a first step toward elucidating how GRK2 regulates MALT1, we sought to identify the specific site(s) within GRK2 that interacts with MALT1. We created a series of expression constructs encoding specific GRK2 fragments and tested the ability of these fragments to inhibit CBM-dependent NF-κB activation. “GRK2 N-term” (AA1-173) includes the N-terminal helix (αN) and the regulator of G-protein signaling homology (RH) domain while “GRK2 ΔαN” (AA 30-C) lacks αN and retains all other domains. Using an NF-κB reporter assay, we found that GRK2 N-term and GRK2 ΔαN each block CBM-dependent NF-κB activation. These results suggest that RH, the only domain retained in both GRK2 fragments, is sufficient to inhibit MALT1. Since the GRK2 RH domain is known to interact with the alpha subunit of guanine-nucleotide binding proteins (Gα), we hypothesized that this protein interaction site may also mediate GRK2 RH binding to MALT1. However, we found that mutations that abrogate GRK2 binding to Gα do not affect GRK2's ability to inhibit CBM-dependent NF-κB activation, suggesting that GRK2 interacts with MALT1 via a different site. We next compared the ability of GRK family members, which each have structurally homologous RH domains, to inhibit CBM-dependent NF-κB activity. We found that, like GRK2, GRK3 inhibits CBM-dependent NF-κB activity while GRK1 does not. We are now comparing RH domain structures for GRK2, GRK3, and GRK1 to identify structural requirements for RH interaction with MALT1. Also, we performed in silico modeling, which predicts that a short stretch of amino acids within GRK2 RH, designated “P1-P2,” interacts with the MALT1 Death Domain. Together, our data suggest that the GRK2 RH domain is sufficient to inhibit MALT1. Ongoing work aims to narrow down the region(s) within the RH that mediates GRK2 binding and inhibition of MALT1. We hope to utilize these analyses to inform the development of a novel class of MALT1 inhibitors for treatment of MALT1-dependent leukemia and lymphoma. Citation Format: Lisa M. Maurer, Matthew Trotta, Jing Cheng, Beibei Bill Chen, Peter C. Lucas, Linda M. McAllister-Lucas. Structural requirements for GRK2-mediated inhibition of the MALT1 proto-oncoprotein [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-13.

Identification of 4-carboxyglutamate residue sites based on position based statistical feature and multiple classification

Glutamic acid is an alpha-amino acid used by all living beings in protein biosynthesis. One of the important glutamic acid modifications is post-translationally modified 4-carboxyglutamate. It has a significant role in blood coagulation. 4-carboxyglumates are required for the binding of calcium ions. On the contrary, this modification can also cause different diseases such as bone resorption, osteoporosis, papilloma, and plaque atherosclerosis. Considering its importance, it is necessary to predict the occurrence of glutamic acid carboxylation in amino acid stretches. As there is no computational based prediction model available to identify 4-carboxyglutamate modification, this study is, therefore, designed to predict 4-carboxyglutamate sites with a less computational cost. A machine learning model is devised with a Multilayered Perceptron (MLP) classifier using Chou’s 5-step rule. It may help in learning statistical moments and based on this learning, the prediction is to be made accurately either it is 4-carboxyglutamate residue site or detected residue site having no 4-carboxyglutamate. Prediction accuracy of the proposed model is 94% using an independent set test, while obtained prediction accuracy is 99% by self-consistency tests.

The phenotype associated with variants in TANGO2 may be explained by a dual role of the protein in ER-to-Golgi transport and at the mitochondria.

TANGO2 variants result in a complex disease phenotype consisting of recurrent crisis-induced rhabdomyolysis, encephalopathy, seizures, lactic acidosis, hypoglycemia, and cardiac arrhythmias. Although first described in a fruit fly model as a protein necessary for some aspect of Golgi function and organization, its role in the cell at a fundamental level has not been addressed. Such studies are necessary to better counsel families regarding treatment options and nutrition management to mitigate the metabolic aspects of the disease. The few studies performed to address the pathway(s) in which TANGO2 functions have led to enigmatic results, with some suggesting defects in membrane traffic while others suggest unknown mitochondrial defects. Here, we have performed a robust membrane trafficking assay on fibroblasts derived from three different individuals harboring TANGO2 variants and show that there is a significant delay in the movement of cargo between the endoplasmic reticulum and the Golgi. Importantly, this delay was attributed to a defect in TANGO2 function. We further show that a portion of TANGO2 protein localizes to the mitochondria through a necessary but not sufficient stretch of amino acids at the amino terminus of the protein. Fibroblasts from affected individuals also displayed changes in mitochondrial morphology. We conclude that TANGO2 functions in both membrane trafficking and in some as yet undetermined role in mitochondria physiology. The phenotype of affected individuals can be partially explained by this dual involvement of the protein.

The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations

Inorganic polyphosphate (polyP) is a morphogenetically active and metabolic energy-delivering physiological polymer that is released from blood platelets. Here, we show that polyP efficiently inhibits the binding of the envelope spike (S)-protein of the coronavirus SARS-CoV-2, the causative agent of COVID-19, to its host cell receptor ACE2 (angiotensin-converting enzyme 2). To stabilize polyP against the polyP-degrading alkaline phosphatase, the soluble polymer was encapsulated in silica/polyP nanoparticles. Applying a binding assay, soluble Na-polyP (sizes of 40 Pi and of 3 Pi units) as well as silica-nanoparticle-associated polyP significantly inhibit the interaction of the S-protein with ACE2 at a concentration of 1 µg/mL, close to the level present in blood. This inhibition is attributed to an interaction of polyP with a basic amino acid stretch on the surface of the receptor binding domain of S-protein. PolyP retains its activity in a flushing solution, opening a new strategy for the prevention and treatment of SARS-CoV-2 infection in the oropharyngeal cavity. The data suggest that supplementation of polyP might contribute to a strengthening of the human innate immunity system in compromised, thrombocytopenic COVID-19 patients.

Driving CARs with alternative navigation tools - the potential of engineered binding scaffolds.

T cells that are genetically engineered to express chimeric antigen receptors (CAR T cells) have shown impressive clinical efficacy against B-cell malignancies. In contrast to these highly potent CD19-targeting CAR T cells, many of those directed against other tumor entities and antigens currently suffer from several limitations. For example, it has been demonstrated that many scFvs used as antigen-binding domains in CARs show some degree of oligomerization, which leads to tonic signaling, T cell exhaustion, and poor performance invivo. Therefore, in many cases alternatives to scFvs would be beneficial. Fortunately, due to the development of powerful protein engineering technologies, also non-immunoglobulin-based scaffolds can be engineered to specifically recognize antigens, thus eliminating the historical dependence on antibody-based binding domains. Here, we discuss the advantages and disadvantages of such engineered binding scaffolds, in particular with respect to their application in CARs. We review recent studies, collectively showing that there is no functional or biochemical aspect that necessitates the use of scFvs in CARs. Instead, antigen recognition can also be mediated efficiently by engineered binding scaffolds, as well as natural ligands or receptors fused to the CAR backbone. Finally, we critically discuss the risk of immunogenicity and show that the extent of nonhuman amino acid stretches in engineered scaffolds-even in those based on nonhuman proteins-is more similar to humanized scFvs than might be anticipated. Together, we expect that engineered binding scaffolds and natural ligands and receptors will be increasingly used for the design of CAR T cells.

Residue-Specific Message Encoding in CD40-Ligand.

SummaryCD40-Ligand (CD40L)-CD40 interaction regulates immune responses against pathogens, autoantigens, and tumor and transplantation antigens. Single amino acid mutations within the 115–155 amino acids stretch, which is responsible for CD40L functions, result in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message that, when decoded by CD40 signaling, induces a specific profile of functions. We observed that every single substitution in the XIgM-related amino acids in the 115–155 41-mer peptide in CD40L selectively altered CD40 signaling and effector functions—cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, survival of B cells, and control of Leishmania infection and anti-leishmanial T cell response—suggesting residue-specific encoding of a distinct set of messages that collectively define CD40L pleiotropy, serve as a target for engineering the ligand to generate superagonists as immunotherapeutic, and implicate the evolutionary diversification of functions among the ligands in a protein superfamily.

A cell-penetrant lactam-stapled peptide for targeting eIF4E protein-protein interactions.

Eukaryotic translation initiation factor 4E (eIF4E) has emerged as a promising cancer therapeutic target due to its role in the initiation of cap-dependent translation, a process that is accelerated during tumorigenesis. To regulate the initiation of cap-dependent translation, eIF4E participates in protein-protein interactions (PPI) with binding partners, 4E-BP1 and eIF4G, which act as an inhibitor and stimulator of translation, respectively. As both of these proteins interact with eIF4E by utilizing a short, α-helical stretch of amino acids, our laboratory has been working to develop helical mimetics of these proteins, in particular 4E-BP1, to inhibit eIF4E PPIs. Herein, we describe our continued efforts in this area and report the development and characterization of a cell-penetrant lactam stapled peptide for targeting cellular eIF4E.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Reduced RNA silencing suppressor activity of onion yellow dwarf virus HC-Pro with N-terminal deletion may be complemented in mixed infection with another potyvirus in garlic

We investigated virus infection rates in Japanese garlic plants and found that a potyvirus, onion yellow dwarf virus (OYDV) was always associated with another potyvirus, leek yellow stripe virus (LYSV). When the OYDV HC-Pro genes from those viral isolates were PCR-amplified and sequenced, we found that all the HC-Pros isolated from Japanese garlic lacked a long stretch of the N-terminal amino acids (~ 100 residues) including the KITC motif, suggesting possible loss of viral aphid transmission. Our phylogenetic analysis revealed that HC-Pros were divided into three major groups and that they did not branch from a single common ancestor, suggesting different mechanisms for generation of the short HC-Pros from the intact ones. Because HC-Pro is a well-known RNA silencing suppressor (RSS), we then analyzed the RSS activity of HC-Pro using the conventional agroinfiltration assay with Nicotiana benthamiana and also with onion, which is a natural host of OYDV. The results demonstrated that the short HC-Pro had reduced RSS activity compared to the long HC-Pro while both type HC-Pros were well expressed in the agroinfiltrated tissues. In addition, the tested OYDV HC-Pros exhibited more efficient RSS activity in onion than in N. benthamiana. We presume that OYDV with reduced RSS activity of HC-Pro may take advantage of mixed infection with LYSV to survive.

Single Receptor reciprocity as message encrypted in CD40-ligand

Abstract Interaction between CD40-Ligand (CD40L) and CD40 results in cellular signaling that regulates immune responses against pathogens, autoantigens, tumors and transplantation antigens. The 115–155 amino acids stretch is responsible for CD40L functions and mutations therein results in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message. These messages are decoded by CD40 signaling and are expressed as a specific profile of functions. Each single substitution in the XIgM-related amino acids in the 115–155 41-mer peptides in CD40L selectively altered CD40 signaling and effector functions- cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, and control of Leishmania infection. There were sets of peptides that triggered reciprocal functions from the same receptor CD40. These results suggest residue-specific encryption of a distinct set of messages. These messages collectively frame CD40L pleiotropy and offer a result-driven ligand manipulation strategy for generating antagonists or superagonists as immunotherapeutics.

Viruses with different genome types adopt a similar strategy to pack nucleic acids based on positively charged protein domains

Capsid proteins often present a positively charged arginine-rich sequence at their terminal regions, which has a fundamental role in genome packaging and particle stability for some icosahedral viruses. These sequences show little to no conservation and are structurally dynamic such that they cannot be easily detected by common sequence or structure comparisons. As a result, the occurrence and distribution of positively charged domains across the viral universe are unknown. Based on the net charge calculation of discrete protein segments, we identified proteins containing amino acid stretches with a notably high net charge (Q > + 17), which are enriched in icosahedral viruses with a distinctive bias towards arginine over lysine. We used viral particle structural data to calculate the total electrostatic charge derived from the most positively charged protein segment of capsid proteins and correlated these values with genome charges arising from the phosphates of each nucleotide. We obtained a positive correlation (r = 0.91, p-value <0001) for a group of 17 viral families, corresponding to 40% of all families with icosahedral structures described to date. These data indicated that unrelated viruses with diverse genome types adopt a common underlying mechanism for capsid assembly based on R-arms.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches – formerly indicated to be involved in CPR:CYP interactions – or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Residue-Specific Message Encryption in CD40-Ligand

CD40-Ligand (CD40L)-CD40 interaction regulates immune responses againstpathogens, autoantigens, tumor and transplantation antigens. Single amino acidmutations within the 115-155 amino acids stretch, which is responsible for CD40L functions, result in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message that, when decoded by CD40 signaling, induces a specific profile of functions. We observed that every single substitution in the XIgM related amino acids in the 115-155 41-mer peptide in CD40L selectively altered CD40 signaling and effector functions- cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, survival of B-cells, and control of Leishmania infection and anti-leishmanial T cell response- suggesting residue-specific encryption of a distinct set of messages that collectively define CD40L pleiotropy, serve as a target for engineering the ligand to generate superagonists as immunotherapeutics and implicate the evolutionary diversification of functions among the ligands in a protein superfamily.

A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations.

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71–82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71–82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71–82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71–82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

Multiple nuclear localization sequences in SRSF4 protein.

SRSF4 is one of the members of serine-/arginine (SR)-rich protein family involved in both constitutive and alternative splicing. SRSF4 is localized in the nucleus with speckled pattern, but its nuclear localization signal was not determined. Here, we have identified nuclear localization signals (NLSs) of SRSF4 by using a pyruvate kinase fusion system. As expected, arginine-/serine (RS)-rich domain of SRSF4 confers nuclear localization activity when it is fused to PK protein. We then further delineated the minimum sequences for nuclear localization in RS domain of SRSF4. Surprisingly, RS-rich region does not always have a nuclear localization activity. In addition, basic amino acid stretches that resemble to classical-type NLSs were identified. These results strongly suggest that SRSF4 protein uses two different nuclear import pathways with multiple NLSs in RS domain.

Characterisation of a novel Emaravirus identified in mosaic‐diseased Eurasian aspen (Populus tremula)

AbstractSince Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic‐diseased Eurasian aspen (Populus tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA‐dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot‐associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT‐PCR in mosaic‐diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic‐diseased Eurasian aspen by virus‐specific RT‐PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic‐disease is graft‐transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft‐inoculated seedlings used as rootstocks as well as in the virus‐infected scions used for graft‐inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic‐associated virus.

Molecular Characterization and Taurine Regulation of Taurine Transporter in Goldfish

AbstractTaurine transporter (TauT) is a sodium‐ and chloride‐dependent transport carrier, and it plays a key role in the transportation of taurine. At present, the many functions of TauT in fish remain obscure. In this study, a cDNA clone for encoding a taurine transporter was isolated from Goldfish Carassius auratus. The nucleotide sequence predicted 625 amino acids with significant homology to the TauT gene of Zebrafish Danio rerio. Hydropathic analysis revealed stretches of hydrophobic amino acids that were indicative of 12 transmembrane domains that were similar to those proposed for other cloned TauT genes. The results of a reverse transcription polymerase chain reaction (RT‐PCR) analysis demonstrated that TauT broadly expressed in all of the selected tissues and developmental stages in Goldfish but with prominent differential expression levels. The expression of TauT in Goldfish has diurnal changes. The highest expression was at 1200 hours, and the lowest was at 2100 hours. Moreover, taurine treatments showed similar stimulatory effects on the expression of TauT in the intestines of Goldfish both in vitro and in vivo. Overall, these results may provide new insights into the regulation of TauT expression in fish, which has potential applications for improving fish dietary formulations.

Mechanistic explanation of structural and functional changes induced by methionine mutation in G-CSF protein

Abstract Potency of a biotherapeutic is influenced by its binding affinity to the designated receptor., This study examines how substitution of methionine residues with Leucine/Alanine in granulocyte colony stimulating factor (G-CSF) brings about changes in its affinity to G-CSF receptor. To this effect, two mutants M1 (M122A, M127A and M138A) and M2 (M122L, M127L and M138L) were designed computationally and their stability and functional attributes were compared with the native GCSF. The simulation results show that although all atom root mean square deviations during 100 ns window were within 2–3 A for the mutants as well as the native G-CSF, yet some local conformational changes were evident. Amino acid residues in the stretch 125–145 exhibited shift in their secondary structure composition from predominantly turn form to coiled and gamma turn forms. In addition, increase in the helical content in the amino acid stretch 60–70 was also observed when compared to native G-CSF. Docking and MMPBSA studies of the GCSF variants with G-CSFR show evidence of increased number of interactions with G-CSFR owing to the observed conformational perturbations in the variants. Experimental data on the stability and the affinity of these variants are in agreement with the in-silico predictions. Thus, we show that careful substitution of key residues in G-CSF potentially presents avenues for improving the stability as well therapeutic prospects of the molecule via enhancement in their affinities to the pertinent receptors.

Nonsynonymous Mutations in Linker-2 of the Pdr5 Multidrug Transporter Identify a New RNA Stability Element.

Analysis of synonymous mutations established that although the primary amino acid sequence remains unchanged, alterations in transcription and translation can result in significant phenotypic consequences. We report the novel observation that a series of nonsynonymous mutations in an unconserved stretch of amino acids found in the yeast multidrug efflux pump Pdr5 increases expression, thus enhancing multidrug resistance. Cycloheximide chase experiments ruled out the possibility that the increased steady-state level of Pdr5 was caused by increased protein stability. Quantitative-RT PCR experiments demonstrated that the mutants had levels of PDR5 transcript that were two to three times as high as in the isogenic wild-type strain. Further experiments employing metabolic labeling of mRNA with 4-thiouracil followed by uracil chasing showed that the half-life of PDR5 transcripts was specifically increased in these mutants. Our data demonstrate that the nucleotides encoding unconserved amino acids may be used to regulate expression and suggest that Pdr5 has a newly discovered RNA stability element within its coding region.