Substitution Of Proline Research Articles

Analysis of Proline Substitutions Reveals the Plasticity and Sequence Sensitivity of Human IAPP Amyloidogenicity and Toxicity.

Pancreatic amyloid formation by the polypeptide IAPP contributes to β-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid in vitro and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P. Incorporation of these proline residues into human IAPP leads to a non-amyloidogenic analogue that is used clinically. The role of the individual proline residues is not understood. We examine the three single and three double proline substitutions in the context of human IAPP. An S28P substitution significantly decreases amyloidogenicity and toxicity, while an S29P substitution has very modest effects despite being an identical replacement just one residue away. The consequences of the A25P substitution are between those of the two Ser to Pro substitutions. Double analogues containing an S28P replacement are less amyloidogenic and less toxic than the IAPPA25PS29P double analogue. Ion mobility mass spectrometry reveals that there is no correlation between the monomer or dimer conformation as reported by collision cross section measurements and the time to form amyloid. The work reveals both the plasticity of IAPP amyloid formation and the exquisite sequence sensitivity of IAPP amyloidogenicity and toxicity. The study highlights the key role of the S28P substitution and provides information that will aid in the rational design of soluble variants of IAPP. The variants studied here offer a system for further exploring features that control IAPP toxicity.

A Mutation in the Mitochondrial Aspartate/Glutamate Carrier Leads to a More Oxidizing Intramitochondrial Environment and an Inflammatory Myopathy in Dutch Shepherd Dogs.

Background:Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed.Objective:A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause.Methods:Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed.Results:Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress.Conclusions:This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.

Critical effect of proline on thermostability of endoglucanase II from Penicillium verruculosum

Thermostability is an important protein property essential for the protein industrial application. Different approaches of rational design are used for protein thermostability improvement. In this research, the effect of proline substitutions on the protein thermostability was tested. Several types of available data on protein characteristics were used for substitution position choosing in order to find those that are critical for thermostability improvement while maintaining the specific activity. The approach used was based on combined analysis of protein structure, structure-function relationship, multiple sequence alignment, and folding free energy. Proline substitutions that were carried out had a critical effect on thermostability of endoglucanase II from Penicillium verruculosum. The most stabilizing substitution S308P provided a 4- and 2.4-fold increase in time of half-life at 70 °C and 80 °C with maintaining the specific activity. Molecular dynamics simulations revealed the observed effects on the protein structure and indicated principal changes in flexibility and hydrogen bonds networks.

Synergistic long-range effects of mutations underlie aggregation propensities of amylin analogues.

The USFDA has approved pramlintide, commercially named Symlin (sIAPP), as adjunctive therapy for type 2 diabetes (T2D). This analogue of the human amylin peptide (hIAPP) has triple proline substitutions typical of the rat isoform (rIAPP). Recently, it was proposed that pramlintide solubility and aggregation resistance might be improved by incorporating further mutations, as S20R, screened from the wild-type porcine isoform (pIAPP), which leads to the variant named sIAPP+. To better elucidate how such properties might be systematically induced in rationally designed analogues, we performed comparative assessments of rIAPP, sIAPP, and sIAPP+ using replica-exchange molecular dynamics (REMD) with an accurate combination of force field Charmm22* and explicit aqueous solvation TIP4P/Ew. Our thermo-structural analyses show that sIAPP exhibits a thermal conversion channel of helices[Formula: see text]-sheets resembling hIAPP. This channel is depleted in rIAPP and is absent in sIAPP+. As a consequence, sIAPP+ presents an overall decrease of β-like secondary structures and an overstabilization of α-helices. Additionally, we observed in rIAPP and sIAPP+ an increase in the backbone RMSF of molecular terminals and the exposed area of key residues. These structural features of sIAPP+ suggest a nonamyloidogenic character, which is corroborated by our judicious estimate of the electrostatic component of the solvation free energy using a generalized Born model, and so it may constitute an alternative strategy to sIAPP as a peptide analogue of hIAPP. Furthermore, our findings confirm that different aggregation propensities of amylin and its analogues are synergistically modulated by long-range effects of key mutations. Graphical Abstract S20R-Pramlintide.

De novo substitutions of TRPM3 cause intellectual disability and epilepsy

The developmental and epileptic encephalopathies (DEE) are a heterogeneous group of chronic encephalopathies frequently associated with rare de novo nonsynonymous coding variants in neuronally expressed genes. Here, we describe eight probands with a DEE phenotype comprising intellectual disability, epilepsy, and hypotonia. Exome trio analysis showed de novo variants in TRPM3, encoding a brain-expressed transient receptor potential channel, in each. Seven probands were identically heterozygous for a recurrent substitution, p.(Val837Met), in TRPM3’s S4–S5 linker region, a conserved domain proposed to undergo conformational change during gated channel opening. The eighth individual was heterozygous for a proline substitution, p.(Pro937Gln), at the boundary between TRPM3’s flexible pore-forming loop and an adjacent alpha-helix. General-population truncating variants and microdeletions occur throughout TRPM3, suggesting a pathomechanism other than simple haploinsufficiency. We conclude that de novo variants in TRPM3 are a cause of intellectual disability and epilepsy.

A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an ∼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.

Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.

B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization.

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large‐scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B‐factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half‐life at 60 °C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 °C (variant K167P). Finally, the presented method utilizing B‐factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.

A KRT16 mutation in the first Chinese pedigree with Pachyonychia congenita and review of the literatures.

Pachyonychia congenita (PC), a rare autosomal dominant disorder, is featured by significant hypertrophic nail, palmoplantar keratoderma, and plantar pain. It is caused by the mutation of KRT6A, KRT6B, KRT6C, KRT16, or KRT17. To identify the gene mutation caused the PC in a Chinese family. Genomic DNA was extracted from peripheral blood samples of five patients and six healthy individuals. Genomic DNA of three patients was sequenced by whole-exome sequencing (WES). Then, exons 6 of KRT16 of all samples were amplified by polymerase chain reaction (PCR), and PCR products were sequenced to identify potential mutations. We identified the proline substitution mutation p.Leu421Pro (c.1262T>C) in the 2B domain of K16 that is associated with PC in a Chinese family. The same mutation was not found in the six healthy individuals of the family. The mutation found in this studyis the first report in China. So far, 25 mutations in KRT16 have been reportedly associated with PC. Twenty-one mutations are located on exon 1, and four mutations on exon 6.

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.

EPSPS duplication and mutation involved in glyphosate resistance in the allotetraploid weed species Poa annua L.

Poa annua is a widespread winter annual weed species in California. Recently, poor control of this species with glyphosate was reported by growers in an almond orchard in California with a history of repetitive glyphosate use. The objectives of this research were to evaluate the level of glyphosate resistance in a developed S4 P. annua line (R) and identify the mechanisms of resistance involved. Whole-plant dose-response experiments confirmed glyphosate resistance in R, which required 18-fold more glyphosate to achieve a 50% growth reduction compared with a susceptible line (S), results that were supported by the lower shikimate accumulation observed in R compared with S. No differences in glyphosate absorption, translocation, or metabolism were observed, suggesting that non-target-site mechanisms of resistance are not involved in the resistance phenotype. A missense single nucleotide polymorphism was observed in EPSPS coding position 106 in R, resulting in a leucine to proline substitution. This polymorphism was observed exclusively in P. supina EPSPS homeologs. A seven-fold increase in the number of copies of EPSPS alleles was observed in R compared with S. We report the first case of glyphosate resistance associated with both EPSPS duplication and target-site mutation at position 106, leading to high levels of glyphosate resistance in the allotetraploid weed species Poa annua L. Data obtained in this research will be useful for the development of diagnostic tools for rapid glyphosate resistance identification, monitoring and containment. © 2018 Society of Chemical Industry.

Identification of a Helical Segment within the Intrinsically Disordered Region of the PCSK9 Prodomain

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a key regulator of lipid metabolism by degrading liver LDL receptors. Structural studies have provided molecular details of PCSK9 function. However, the N-terminal acidic stretch of the PCSK9 prodomain (Q31–T60) has eluded structural investigation, since it is in a disordered state. The interest in this region is intensified by the presence of human missense mutations associated with low and high LDL-c levels (E32K, D35Y, and R46L, respectively), as well as two posttranslationally modified sites, sulfated Y38 and phosphorylated S47. Herein we show that a segment within this region undergoes disorder-to-order transition. Experiments with acidic stretch-derived peptides demonstrated that the folding is centered at the segment Y38–L45, which adopts an α-helix as determined by NMR analysis of free peptides and by X-ray crystallography of peptides in complex with antibody 6E2 (Ab6E2). In the Fab6E2–peptide complexes, the structured region features a central 2 1/4-turn α-helix and encompasses up to 2/3 of the length of the acidic stretch, including the missense mutations and posttranslationally modified sites. Experiments with helix-breaking proline substitutions in peptides and in PCSK9 protein indicated that Ab6E2 specifically recognizes the helical conformation of the acidic stretch. Therefore, the observed quantitative binding of Ab6E2 to native PCSK9 from various cell lines suggests that the disorder-to-order transition is a true feature of PCSK9 and not limited to peptides. Because the helix provides a constrained spatial orientation of the missense mutations and the posttranslationally modified residues, it is probable that their biological functions take place in the context of an ordered conformational state.

Alternative Mutations and Isoform Dysregulation in MYD88 in Waldenstrom's Macroglobulinemia

BackgroundMutations in MYD88 are highly recurring in Waldenstrom's Macroglobulinemia (WM) patients and are important for establishing the diagnosis of WM. The most common mutation in MYD88 is c.978T>C resulting a proline substitution for leucine at amino acid position 265 (p.Leu265Pro). Both allele specific PCR (AS-PCR) and clinical diagnostic next generation sequencing (NGS) panels are used to detect mutated MYD88, though they differ in sensitivity and scope. In this study we screened 734 patients with WM by AS-PCR for MYD88 c.978T>C MYD88 followed by Sanger sequencing to clarify negative results for non-MYD88 p.Leu265Pro mutations and compared the findings to clinical NGS panel data from the same biopsy when available. We also investigated MYD88 isoform dysregulation and isoform specific effects of the observed mutations that may impact mutated MYD88 regulation which has not been previously studied in WM.MethodsDNA from CD19-selected bone marrow mononuclear cells (BMMC) of 734 WM patients were used for the MYD88 c.978T>C AS-PCR assay previously described by us (Xu et al, Blood 2013). For patients wild-type for MYD88 c.978T>C by AS-PCR, Sanger sequencing of the open reading frame of MYD88 was performed for both DNA and RNA simultaneously isolated from CD19-selected BMMC. DNA was also used to validate the presence of c.978T>C by Sanger. Findings were compared to 222/734 (30.2%) patients who also underwent illumina miSeq based targeted next generation sequencing on a clinical diagnostic platform using unselected BMMC. NGS isoform specific expression estimates were calculated using Salmon for 77 WM patients and 34 healthy donors (Hunter et al, Blood 2016).Results688/734 (93.7%) WM patients tested positive for the c.978T>C mutation. To confirm these results, Sanger sequencing at the DNA level covering the c.978T>C mutation was performed in 361/688 (52.5%) patients confirming the presence of the mutation in all cases. These Sanger studies revealed that one patient had two somatic mutations in addition to c.978T>C. Of the 46/734 (6.3%) that were wild-type by AS-PCR, 18 had cDNA available to screen for alternative MYD88 mutations. Of these, 13/18 (72.2%) were confirmed to be truly wild type for MYD88, and 5/18 (27.8%) harbored alternative MYD88 mutations making up 0.7% of the study population. Taken together 693/734 (94.4%) of patients were found to harbor somatic MYD88 mutations. Of the 222 patients form whom matching NGS panel data was available, the finding between the NGS and AS-PCR studies were largely concordant. The only discrepancies observed were 69 (31.1%) cases where targeted NGS gave false negative results for c.978T>C but was detected by AS-PCR. Of the four patients with alternative MYD88 mutations, one patient had a dinucleotide substitution that also resulted in p.Leu265Pro but tested as wild-type by AS-PCR, two patients each had one previously documented mutation (either pVal217Phe or p.Ser243Asn) and one patient had a mutation that was synonymous at the protein level (p.Phe277Phe). The patient with the two novel mutations in addition to c.978T>C had a mutation in the polypyrimidine track leading to the final exon and one resulting in p.Gly259Gly in the primary transcript but presents as a highly disruptive p.Val199Glu in the shorter regulatory isoforms. This is similar to c.978T>C which presents as p.L265P in the primary transcripts but acts as a stop loss in the shorter isoforms. We therefore looked for evidence of isoform level dysregulation in MYD88 using RNASeq and found highly significant and distinctive MYD88 isoform signatures for MYD88 mutant, MYD88 wild-type and healthy donor samples (Figure 1).ConclusionsUsing CD19-selected BMMC, MYD88 c.978T>C (p.Leu265Pro) was found in 93.7% of 734 patients, while non-c.978T>C mutations were present in <1% of WM population. Discordance with targeted NGS is common, with a false negative rate of 31%. Distinct isoform usage patterns and isoform specific mutational effects among MYD88 mutated patients was observed, highly suggestive of a functional role for MYD88 splicing in WM pathogenesis. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; BMS: Research Funding; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment

Tau aggregation is a hallmark of a group of neurodegenerative diseases termed Tauopathies. Reduction of aggregation-prone Tau has emerged as a promising therapeutic approach. Here, we show that an anti-aggregant Tau fragment (F3ΔKPP, residues 258–360) harboring the ΔK280 mutation and two proline substitutions (I277P & I308P) in the repeat domain can inhibit aggregation of Tau constructs in vitro, in cultured cells and in vivo in a Caenorhabditis elegans model of Tau aggregation. The Tau fragment reduced Tau-dependent cytotoxicity in a N2a cell model, suppressed the Tau-mediated neuronal dysfunction and ameliorated the defective locomotion in C. elegans. In vitro the fragment competes with full-length Tau for polyanionic aggregation inducers and thus inhibits Tau aggregation. Our combined in vitro and in vivo results suggest that the anti-aggregant Tau fragment may potentially be used to address the consequences of Tau aggregation in Tauopathies.

Structure-Guided Redesign Improves NFL HIV Env Trimer Integrity and Identifies an Inter-Protomer Disulfide Permitting Post-Expression Cleavage.

Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P. This P improved trimer integrity compared to I559P in selected properties. Next, we screened 15 structure-guided potential cysteine pairs in gp140 and found that A501C-L663C (“CC2”) forms an inter-protomer disulfide bond that demonstrably increased NFL trimer thermostability. We combined these two approaches with trimer-derived substitutions, coupled with glycine substitutions at helix-to-coil transitions, developed by our group. To increase the exposure of the fusion peptide (FP) N-terminus, we engineered an enterokinase (EK) cleavage site upstream of the FP for controlled post-expression cleavage. In combination, the redesigns resulted in highly stable and homogeneous NFL mimics derived from different clades. Following recombinant EK cleavage, the NFL trimers retained covalent linkage, maintaining a native-like structure while displaying enhanced stability and favorable antigenic features. These trimers also displayed increased exposure of neutralizing epitopes in the FP and gp120/gp41 interface, while retaining other neutralizing epitopes and occluding non-neutralizing elements. This array of Env-structure-guided designs reveals additional interactive regions in the prefusion state of the HIV Env spike, affording the development of novel antigens and immunogens.

Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L.

Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be(-KX)) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.

Wnt5a induces ROR1 to recruit DOCK2 to activate Rac1/2 in chronic lymphocytic leukemia

AbstractReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) that can serve as a receptor for Wnt5a, which can promote leukemia cell migration, proliferation, and survival. We found Wnt5a could induce ROR1 to complex with DOCK2 (dedicator of cytokinesis 2) and induce activation of Rac1/2; these effects could be blocked by cirmtuzumab, a humanized anti-ROR1 monoclonal antibody. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to induce activation of Rac1/2 or enhance CLL cell proliferation. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for Wnt5a to induce ROR1 to complex with DOCK2 and activate Rac1/2 in the CLL cell-line MEC1. We introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at potential binding sites for the Src-homology 3 domain of DOCK2. In contrast to wild-type ROR1, or other ROR1 P→A variants, ROR1P808A was unable to recruit DOCK2 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P→A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1P808A did not have a growth advantage over MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in patients who have CLL cells that express high levels of ROR1.

Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide β-Hairpin.

Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Coaggregation of heterogeneous peptide species can occur, however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP106-126) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of nonhomologous protein assembly as they coaggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only forms pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic β-hairpin conformation. Further, when PrP106-126 was combined with non-amyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing β-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic β-hairpin structure, it did so in the presence of PrP106-126 and underwent a conformational transition to β-sheet in solution. We also find that PrP106-126 forms hetero-oligomers with the IAPP8-20 fragment but not with the "aggregation hot spot" IAPP20-29 fragment. PrP106-126 apparently induces IAPP into a β-hairpin structure within the PrP:IAPP heterodimer complex and then, through ligand exchange, catalytically creates the amyloidogenic β-hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model that provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.

Abstract 3492: Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on breast cancer, but not on virtually all normal adult tissues. Studies have found that high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival compared to that of patients with tumors that had low expression of ROR1 (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). Analysis of ROR1-immune precipitates of primary breast cancer patient-derived-xenograft (PDX) lysates via immunoblot analyses revealed that ROR1 was associated with DOCK1 (Dedicator of Cytokinesis 1, also known as DOCK180) in breast cancer PDX tumors. DOCK1 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 that is expressed in breast cancer PDX tumors or cell lines. DOCK1 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK1 in PDX cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a. Wnt5a could induce ROR1 to complex with DOCK1 and cause activation of Rac1; this effect could be inhibited by silencing DOCK1 in PDX cells. Furthermore, these effects of Wnt5a on PDX cells also could be blocked by treatment of the PDX cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. We corroborated these findings using the breast cancer cell-line MCF7, which also expresses DOCK1, but does not express ROR1. MCF7 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK1 interactions. We confirmed that DOCK1 complexes with ROR1 in response to Wnt5a in MCF7-ROR1 cells. We find that silencing DOCK1 specifically impaired the capacity of Wnt5a to enhance growth of MCF7-ROR1 cells in vitro. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK1 and activate Rac1 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P-&amp;gt;A mutants, ROR1P(808)A had impaired capacity to recruit DOCK1 to ROR1 in response to Wnt5a. Moreover, unlike MCF7 cells transfected with wild-type ROR1 or ROR1 with P-&amp;gt;A substitutions at positions 784, 826, or 841, MCF7 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MCF7 cells that do not express ROR1. This study reveals that the recruitment of DOCK1 may be critical for the capacity of Wnt5a to enhance breast cancer cell growth, which may contribute to the observed increased tendency for disease progression in breast cancer patients who have tumors that express high-levels of ROR1. Citation Format: Md Kamrul Hasan, Victoria Tripple, George F. Widhopf, Suping Zhang, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3492.

Engineering Clostridium absonum 7α-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and ΔΔG Prediction.

Enhancing thermostability of the 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) is beneficial to its industrial application broadly. For protein engineering to enhance thermostability the nonrational strategy, directed evolution, has been applied in obtaining more stable proteins through error-prone PCR or DNA rearrangement generating random mutations. However, the successful application of directed evolution needs to build a large mutant library. Site-directed mutations of CA 7α-HSDH had been performed to probe the relationship between the compactness increasing and thermostability enhancing. Although most of the mutations in β-sheet core predicted by MAESTRO became more stable than wild type, unfortunately, all the mutations suffered dramatic activity loss. The main objective of this study was to verify effects of the mutations in helices selected from the predicting results through MAESTRO on thermostability improving of CA 7α-HSDH. Seven mutants, S22L, P124L, A125L, N171L, A195Q, L197E and Y259E were synthesized and verified through DNA sequencing in Sangon Biotech (Sangon, Shanghai, China). The two mutants, A104P and G105P were prepared by over-lapping PCR. The GST-fusion expression vector, pGEX-6p-1 (GE Healthcare), was used for protein expression with restriction sites BamH I and Not I. Thermostability was measured by circular dichroism (CD) spectrometer (MOS-450, BioLogic Inc). All the enzymes were diluted in PBS (pH 7.3, 10 mM) to OD222 value between 0.8 and 1, and temperature varied from 20°C to 95°C. Activity of enzyme was assayed by measuring the production of NADPH by UV-visible spectrophotometer at 340 nm. The activity assay was performed in 2 mL reaction mixture which contained PBS (pH 7.3, 10 mM), NADP+ (0.5 mM) and taurocholic acid (TCA) at 25°C. Based on unfolding free energy changes (ΔΔG) prediction seven mutations of Clostridium absonum (CA) 7α-HSDH were selected and experimentally verified, and these mutants fitted three-state denaturation model well, among which S22L located in the αA possessed the greatest Tm N→I increase (> 8°C). Mutants P124L, L197E, N171L and Y259E also became more stable than wild type CA 7α-HSDH with different ranges. Meanwhile, thermostability of the two mutants, A104P and G105P (in the coil between βD and αD) resulting from the proline substitution method decreased significantly. Enzyme activity assays indicated that mutant L197E located in αF maximally maintained 28.7% of catalytic efficiency, and activity of the five mutants, P124L, A125L, N171L, A104P and G105P cannot be detected. Although all the mutants' activities decreased, the mutant L197E with the maximum activity retain suggested that the loop structure (residues 194 to 211) may be the favored candidate sites to enhance thermostability. In addition, CA 7α-HSDH may suffer structural destruction resulting from the proline substitution in A104 and G105.