scholarly journals Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

2017 ◽  
Vol 114 (9) ◽  
pp. 2265-2270 ◽  
Author(s):  
Justin R. Klesmith ◽  
John-Paul Bacik ◽  
Emily E. Wrenbeck ◽  
Ryszard Michalczyk ◽  
Timothy A. Whitehead
Keyword(s):  
Surface Display ◽  
Protein Solubility ◽  
Fitness Landscapes ◽  
Yeast Surface Display ◽  
Crystallographic Structure ◽  
Missense Mutations ◽  
Classification Models ◽  
Wild Type ◽  
Trade Offs ◽  
And Function

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4700-4706 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Marie-Claude Thia ◽  
Joseph A. Trapani
Keyword(s):  
Functional Analysis ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Autosomal Recessive ◽  
Missense Mutations ◽  
Wild Type ◽  
Lytic Function ◽  
Functional Defect ◽  
Target Cell Membrane ◽  
And Function ◽  
Prf1 Gene

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes

2018 ◽  
Vol 475 (12) ◽  
pp. 2107-2125 ◽  
Author(s):  
Nishad Matange ◽  
Swapnil Bodkhe ◽  
Maitri Patel ◽  
Pooja Shah
Keyword(s):  
Drug Resistance ◽  
Structural Stability ◽  
Dihydrofolate Reductase ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Resistance Mutations ◽  
Acquired Drug Resistance ◽  
Trade Offs ◽  
And Function

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases

2013 ◽  
Vol 89 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Marija Blazic ◽  
Gordana Kovacevic ◽  
Olivera Prodanovic ◽  
Raluca Ostafe ◽  
Marija Gavrovic-Jankulovic ◽  
...  
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Wild Type ◽  
Purification And Characterization ◽  
Yeast Surface

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3646-3653 ◽  
Author(s):  
Gabriella Kunz ◽  
Ann-Kristin Öhlin ◽  
Antonella Adami ◽  
Bengt Zöller ◽  
Peter Svensson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Thromboembolic Disease ◽  
Missense Mutations ◽  
Wild Type ◽  
Michaelis Constant ◽  
Complementary Dna ◽  
Naturally Occurring ◽  
Cofactor Activity ◽  
And Function

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% ± 5%, P < .001), Pro477Ser (76.8% ± 1%, P < .001), Pro483Leu (82.1% ± 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (Km) for wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P < .0001) Km, 2967 ±  283 nM, and 2342 ± 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.

scholarly journals A mutant p53/Hif1α/miR-30d axis reprograms the secretory pathway promoting the release of a prometastatic secretome

Cell Stress ◽  
2020 ◽  
Vol 4 (11) ◽  
pp. 261-264
Author(s):  
Valeria Capaci ◽  
Fiamma Mantovani ◽  
Giannino Del Sal
Keyword(s):  
Secretory Pathway ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Wild Type ◽  
Metastatic Colonization ◽  
Non Invasive ◽  
Xenograft Models ◽  
Non Coding Rnas ◽  
Cellular Pathways ◽  
And Function

TP53 missense mutations are frequent driver events during tumorigenesis. The majority of TP53 mutations are missense and occur within the DNA binding domain of p53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the tumor suppressive functions of the wild-type (wt-p53) form, but can also acquire novel oncogenic features fostering tumor growth, metastasis and chemoresistance. Mut-p53 affects fundamental cellular pathways and functions through different mechanisms, a major one being the alteration of gene expression. In our recent work (Capaci et al., 2020, Nat Commun) we found that mut-p53, via miR-30d, modifies structure and function of the Golgi apparatus (GA) and induces increased rate of trafficking. This culminates in the release of a pro-malignant secretome, which is capable of remodeling the tumor microenvironment (TME), to increase stiffness of the extracellular matrix (ECM), favouring metastatic colonization, as shown by cell-based assays and experiments of metastatic niche preconditioning in mouse xenograft models. This study provides new insights into the mechanisms by which mut-p53, through induction of non-coding RNAs, can exert pro-tumorigenic functions in a non-cell-autonomous fashion, and highlights potential non-invasive biomarkers and therapeutic targets to treat tumors harboring mut-p53 (Figure 1).

scholarly journals Engineered IL-10 variants elicit potent immunomodulatory effects at low ligand doses

2020 ◽  
Vol 13 (649) ◽  
pp. eabc0653 ◽  
Author(s):  
Claire Gorby ◽  
Junel Sotolongo Bellón ◽  
Stephan Wilmes ◽  
Walid Warda ◽  
Elizabeth Pohler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Energy Homeostasis ◽  
Surface Display ◽  
Interleukin 10 ◽  
Cytolytic Activity ◽  
Yeast Surface Display ◽  
Receptor Complex ◽  
Wild Type ◽  
Clinical Benefits

Interleukin-10 (IL-10) is a dimeric cytokine with both immunosuppressive and immunostimulatory activities; however, IL-10–based therapies have shown only marginal clinical benefits. Here, we explored whether the stability of the IL-10 receptor complex contributes to the immunomodulatory potency of IL-10. We generated an IL-10 mutant with enhanced affinity for its IL-10Rβ receptor using yeast surface display. Compared to the wild-type cytokine, the affinity-enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes and triggered greater STAT1 and STAT3 activation in human monocytes and CD8+ T cells. These effects, in turn, led to more robust induction of IL-10–mediated gene expression programs at low ligand concentrations in both human cell subsets. IL-10–regulated genes are involved in monocyte energy homeostasis, migration, and trafficking and in CD8+ T cell exhaustion. At nonsaturating doses, IL-10 did not induce key components of its gene expression program, which may explain its lack of efficacy in clinical settings. Our engineered IL-10 variant showed a more robust bioactivity profile than that of wild-type IL-10 at low doses in monocytes and CD8+ T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with wild-type IL-10. Our study provides insights into how IL-10 receptor complex stability fine-tunes IL-10 biology and opens new opportunities to revitalize failed IL-10 therapies.

scholarly journals Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1)

2020 ◽  
Vol 77 (1) ◽  
Author(s):  
Wai L. Lau ◽  
Bradley Pearce ◽  
Heather Malakian ◽  
Iyoncy Rodrigo ◽  
Dianlin Xie ◽  
...  
Keyword(s):  
Immune Response ◽  
Drug Discovery ◽  
Surface Display ◽  
Kinase Domain ◽  
Yeast Surface Display ◽  
Valuable Insight ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Structure Based Drug Design ◽  
Yeast Surface

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular kinase that plays an important role in modulating tumor immune response and thus is an attractive target for drug discovery. Crystallization of the wild-type HPK1 kinase domain has been hampered by poor expression in recombinant systems and poor solubility. In this study, yeast surface display was applied to a library of HPK1 kinase-domain variants in order to select variants with an improved expression level and solubility. The HPK1 variant with the most improved properties contained two mutations, crystallized readily in complex with several small-molecule inhibitors and provided valuable insight to guide structure-based drug design. This work exemplifies the benefit of yeast surface display towards engineering crystallizable proteins and thus enabling structure-based drug discovery.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Beyond Bias

2020 ◽  
pp. 736-753
Author(s):  
Chelsea Barabas
Keyword(s):  
Best Practices ◽  
Criminal Law ◽  
Criminal Justice Reform ◽  
Trade Offs ◽  
System P ◽  
Accuracy Measures ◽  
Criminal Legal System ◽  
And Function ◽  
Role And Function

This chapter discusses contemporary debates regarding the use of artificial intelligence as a vehicle for criminal justice reform. It closely examines two general approaches to what has been widely branded as “algorithmic fairness” in criminal law: the development of formal fairness criteria and accuracy measures that illustrate the trade-offs of different algorithmic interventions; and the development of “best practices” and managerialist standards for maintaining a baseline of accuracy, transparency, and validity in these systems. Attempts to render AI-branded tools more accurate by addressing narrow notions of bias miss the deeper methodological and epistemological issues regarding the fairness of these tools. The key question is whether predictive tools reflect and reinforce punitive practices that drive disparate outcomes, and how data regimes interact with the penal ideology to naturalize these practices. The chapter then calls for a radically different understanding of the role and function of the carceral state, as a starting place for re-imagining the role of “AI” as a transformative force in the criminal legal system.

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