Substitution Of Leucine Research Articles

The inheritance pattern for the dwarf phenotype in hybrids from crosses among sunflower lines differing in alleles of the Rht1 locus

Background. Sunflower seed production is based on utilization of the heterosis effect, manifesting itself in improving both yield and plant height in hybrids. Short-stemmed lines need to be used to develop commercial hybrids with an optimum height. Molecular bases of the trait manifestation in the dwarf lines from VIR’s sunflower genetic collection have not yet been studied. Materials and methods. The material included 27 short-stemmed and 10 tall sunflower lines from VIR’s genetic collection, as well as the F1 and F2 hybrid generations derived from crossing the tall (VIR 340) and dwarf (VIR 171) genotypes. The parental lines and hybrids were phenotyped for plant height, leaf number, and internode length. Genotyping for the Rht1 locus (HaDella1 candidate gene), encoding the negative regulator of the gibberellin response, the DELLA protein, was performed using the developed CAPS marker. Results. The average plant height in the VIR 340 line over a three-year study was 162 cm, the number of leaves 29, and the internode length 6 cm. The VIR 171 line demonstrated the plant height of 66 cm, leaf number of 24, and internode length of 2.8 cm. The F1 hybrids were uniform, with the height of 180–190 cm, that indicated the dominance of the long stem trait. Analyzing the segregation in the F2 hybrid generation led to an assumption admitting the digenic control of the dwarf phenotype in the VIR 171 line. The CAPS marker G-D-1/ Bmt I was developed to identify a missense mutation T>C in the first exon of the HaDella1 gene, which results in the substitution of leucine with proline in the DELLA motif. Using the marker, the mutant Rht1 allele was identified in the VIR 171 and VIR 434 dwarf lines, similar in their origin and phenotype. The results of validation in the F2 hybrid population (VIR 340 × VIR 171) confirmed the efficiency of the G-D-1 / Bmt I marker for selecting dwarf genotypes homozygous for the Rht1 mutant allele.

Dopaminergic neurodegeneration in Gerstmann-Sträussler-Scheinker (P102L) disease: insights from imaging and pathological examination.

Gerstmann-Sträussler-Scheinker (GSS) disease is an inherited prion disease characterized by dementia, cerebellar ataxia, and painful sensory disturbances. GSS is pathologically defined by the presence of amyloid plaques comprised of prion protein predominantly localized in the cerebral cortex, cerebellar cortex, and basal ganglia, resulting from mutations in the prion protein gene. This study investigated five cases of GSS P102L [GSS caused by a leucine (L) substitution of proline (P) at position 102 of the prion protein gene] with L-dopa-resistant extrapyramidal symptoms and reduced dopamine transporter single-photon emission computed tomography (DAT-SPECT) uptake. Clinical findings revealed diverse manifestations, with all cases exhibiting parkinsonism, and four patients had a vertical gaze palsy. Notably, all patients showed reduced striatal DAT-SPECT uptake, indicating neurodegeneration of the nigrostriatal system. Autopsy findings in one case confirmed prion protein plaques and dopaminergic neuron loss in the substantia nigra of a patient with GSS P102L. Additionally, reduced DAT immunostaining was observed in the putamen compared with a control. While previous studies have identified reduced DAT-SPECT and positron emission tomography uptake in Creutzfeldt-Jakob disease and fatal familial insomnia owing to nigrostriatal neurodegeneration induced by abnormal prion protein deposition, similar phenomena in GSS P102L have not been reported. This study provides support for a correlation between abnormal prion protein deposition and nigrostriatal system degeneration in GSS P102L. Our results reveal the importance of considering GSS P102L in cases of atypical Parkinsonism and abnormal DAT-SPECT results, which would serve as a valuable indicator for subsequent prion genetic testing.

Case report: two novel PPARG pathogenic variants associated with type 3 familial partial lipodystrophy in Brazil

Introduction and aimType 3 Familial Partial Lipodystrophy (FPLD3) is a rare metabolic disease related to pathogenic PPARG gene variants. FPLD3 is characterized by a loss of fatty tissue in the upper and lower limbs, hips, and face. FPLD3 pathophysiology is usually associated with metabolic comorbidities such as type 2 diabetes, insulin resistance, hypertriglyceridemia, and liver dysfunction. Here, we clinically and molecularly characterized FPLD3 patients harboring novel PPARG pathogenic variants.Materials and methodsLipodystrophy-suspected patients were recruited by clinicians from an Endocrinology Reference Center. Clinical evaluation was performed, biological samples were collected for biochemical analysis, and DNA sequencing was performed to define the pathogenic variants associated with the lipodystrophic phenotype found in our clinically diagnosed FPLD subjects. Bioinformatics predictions were conducted to characterize the novel mutated PPARγ proteins.ResultsWe clinically described FPLD patients harboring two novel heterozygous PPARG variants in Brazil. Case 1 had the c.533T > C variant, which promotes the substitution of leucine to proline in position 178 (p.Leu178Pro), and cases 2 and 3 had the c.641 C > T variant, which results in the substitution of proline to leucine in the position 214 (p.Pro214Leu) at the PPARγ2 protein. These variants result in substantial conformational changes in the PPARγ2 protein.ConclusionTwo novel PPARG pathogenic variants related to FPLD3 were identified in a Brazilian FPLD cohort. These data will provide new epidemiologic data concerning FPLD3 and help understand the genotype-phenotype relationships related to the PPARG gene.

Enhancing Selective Antimicrobial and Antibiofilm Activities of Melittin through 6-Aminohexanoic Acid Substitution.

Leucine residues are commonly found in the hydrophobic face of antimicrobial peptides (AMPs) and are crucial for membrane permeabilization, leading to the cell death of invading pathogens. Melittin, which contains four leucine residues, demonstrates broad-spectrum antimicrobial properties but also significant cytotoxicity against mammalian cells. To enhance the cell selectivity of melittin, this study synthesized five analogs by replacing leucine with its structural isomer, 6-aminohexanoic acid. Among these analogs, Mel-LX3 exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, Mel-LX3 displayed significantly reduced hemolytic and cytotoxic effects compared to melittin. Mechanistic studies, including membrane depolarization, SYTOX green uptake, FACScan analysis, and inner/outer membrane permeation assays, demonstrated that Mel-LX3 effectively permeabilized bacterial membranes similar to melittin. Notably, Mel-LX3 showed robust antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Furthermore, Mel-LX3 effectively inhibited biofilm formation and eradicated existing biofilms of MDRPA. With its improved selective antimicrobial and antibiofilm activities, Mel-LX3 emerges as a promising candidate for the development of novel antimicrobial agents. We propose that the substitution of leucine with 6-aminohexanoic acid in AMPs represents a significant strategy for combating resistant bacteria.

BnaC03.BIN2 regulates plant height by affecting the main inflorescence length and first effective branch height in Brassica napus L.

Rapeseed (Brassica napus L.) is one of the main oil crops in the world, and increasing its yield is of great significance for ensuring the safety of edible oil. Presently, improving rapeseed plant architecture is an effective way to increase rapeseed yield with higher planting density. However, the regulatory mechanism of rapeseed plant architecture is poorly understood. In this study, a dwarf rapeseed mutant dwarf08 (df08) is obtained by ethyl methane sulfonate (EMS)-mutagenesis. The decrease in plant height of df08 is mainly caused by the reduction in main inflorescence length and first effective branch height and controlled by a single semi-dominant gene. The hybrid plants (F1) show a semi-dwarf phenotype. Through map-based cloning and transgenic assay, we confirm that the nonsynonymous single nucleotide variant (SNV) (C to T) in BnaC03.BIN2, which is homologous with Arabidopsis (Arabidopsis thaliana) BIN2, is responsible for the dwarfism of df08. BnaC03.BIN2 interacts with BnaBZR1/BES1 and involves in brassinosteroids (BRs) signal transduction. Proline to Leucine substitution in 284 (P284L) enhances the protein stability of BnaC03.bin2-D, disrupts BRs signal transduction and affects the expression of genes regulating cell division, leading to dwarfism of df08. This study provides a new insight for the mechanism of rapeseed plant height regulation and creates an elite germplasm that can be used for genetic improvement of rapeseed architecture.

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

A First Reported Combination of Exon 18 Missense (G719X) Mutation and Exon 20 Insertion Mutation (Exon20ins) Of Epithelial Growth Factor Receptor in A Patient with Non-Small Cell Lung Carcinoma

The majority of non-small cell lung cancer (NSCLC) associated with epidermal growth factor receptor (EGFR) mutations involve exon 19 deletion mutations and exon 21-point mutation L858R (arginine for leucine substitution at amino acid 858). G719X point mutation (substitutions of the glycine at position 719 to other residues) in exon 18 and insertion in exon 20 are less commonly detected. However, the combination of the latter two mutations has never been reported. We report the first case of metastatic NSCLC with EGFR mutation harboring both exon 18 missense point mutation (G719X) and exon 20 insertion (exon20Ins).

Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

Dietary Amino Acid Composition and Glycemic Biomarkers in Japanese Adolescents.

Protein intake reportedly increases the risk of diabetes; however, the results have been inconsistent. Diabetes in adulthood may be attributed to early life dietary amino acid composition. This study aimed to investigate the association between amino acid composition and glycemic biomarkers in adolescents. Dietary intake was assessed using a food frequency questionnaire, and fasting glucose and insulin levels were measured in 1238 eighth graders. The homeostatic model assessment (HOMA) indices (insulin resistance and β-cell function) were calculated. Anthropometrics were measured and other covariates were obtained from a questionnaire. Amino acid composition was isometric log transformed according to the compositional data analysis, which was used as explanatory variables in multivariate linear regression models for glucose, insulin, and HOMA indices. Only the association between glucose and leucine was significant. In replacement of other amino acids with leucine, an increase of 0.1% of total amino acids correlated with a lower glucose level (-1.02 mg/dL). One-to-one substitution of leucine for isoleucine or methionine decreased glucose (-2.98 and -2.28 mg/dL, respectively). Associations with other biomarkers were not observed. In conclusion, compositional data analysis of amino acids revealed an association only with glucose in adolescents; however, the results of this study should be verified in other populations.

Smooth pigweed (Amaranthus hybridus) and unresolved Amaranthus spp. from Brazil resistant to glyphosate exhibit the EPSPS TAP-IVS substitution

AbstractThe presence of glyphosate-resistant smooth pigweed (Amaranthus hybridus L.) biotypes has increased in southern Brazil in recent years, presenting the triple amino acid substitution TAP-IVS in 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) previously found in Argentina. Some of these biotypes have morphological characteristics of A. hybridus and redroot amaranth (Amaranthus retroflexus L.). The present study aimed to identify, through molecular markers, the herbicide-resistant species of Amaranthus from Brazil that have the TAP-IVS substitution and to analyze the occurrence of pollen-mediated gene flow (PMGF) as the source of the TAP-IVS substitution in these biotypes. Six biotypes were evaluated using internal transcribed spacer (ITS) sequences, of which two (AMACHY-S and CAMAQ-R) were molecularly classified as A. hybridus and four (AMACRET-S, AMACVI-S, ARRGR-R, and SAOJER-R) were unclassified. Interestingly, all the glyphosate-resistant biotypes (ARRGR-R, SAOJER-R, and CAMAQ-R) had the TAP-IVS substitution and an increase in EPSPS relative copy number; however, only CAMAQ-R was confirmed as A. hybridus. Although the biotypes ARRGR-R and SAOJER-R are closely related to A. hybridus and green pigweed (Amaranthus powellii S. Watson), their species identity could not be resolved. The biotype SAOJER-R also was resistant to acetolactate synthase (ALS)-inhibiting herbicides due to a tryptophan to leucine substitution at position 574 in ALS. The evaluation of 119,746 seedlings in an intraspecific hybridization study of A. hybridus indicated an outcrossing frequency of 0.09%. In contrast, an absence of interspecific hybridization (A. hybridus × unclassified biotype, AMACVI-S) was found after screening 111,429 offspring. Unclassified biotypes might be derived from one or more ancient hybridization events and subsequently evolved the triple mutation independently. Alternatively, such biotypes could have evolved from recent hybridization events, which occur at a frequency below the level of detection in our study.

Analysis of random mutations in Salmonella Gallinarum dihydropteroate synthase conferring sulfonamide resistance.

In bacteria and primitive eukaryotes, sulfonamide antibiotics block the folate pathway by inhibiting dihydropteroate synthase (FolP) that combines para-aminobenzoic acid (pABA) and dihydropterin pyrophosphate (DHPP) to form dihydropteroic acid (DHP), a precursor for tetrahydrofolate synthesis. However, the emergence of resistant strains has severely compromised the use of pABA mimetics as sulfonamide drugs. Salmonella enterica serovar Gallinarum (S. Gallinarum) is a significant source of antibiotic-resistant infections in poultry. Here, a sulfonamide-resistant FolP mutant library of S. Gallinarum was generated through random mutagenesis. Among resistant strains, substitution of amino acid Arginine 171 with Proline (R171P) in the FolP protein conferred the highest resistance against sulfonamide. Substitution of Phe28 with Leu or Ile (F28L/I) led to modest sulfonamide resistance. Structural modeling indicates that R171P and Phenylalanine 28 with leucine or isoleucine (F28L/I) substitution mutations are located far from the substrate-binding site and cause insignificant conformational changes in the FolP protein. Rather, in silico studies suggest that the mutations altered the stability of the protein, potentially resulting in sulfonamide resistance. Identification of specific mutations in FolP that confer resistance to sulfonamide would contribute to our understanding of the molecular mechanisms of antibiotic resistance.

A Novel β-Globin Variant, Hb Raklev [β 75(E19) HBB:c.227T > A (Leu→Gln)

We present a new hemoglobin variant, Hb Raklev, characterized by the substitution of leucine with glutamine at position 75 in the β-globin chain. This variant was discovered inadvertently during an HbA1c evaluation using high performance liquid chromatography in a symptomless 54-year-old Caucasian woman, with the same variant also identified in her 16-year-old daughter. Purification of the hemoglobin revealed possibly diminished 2,3-bisphosphoglycerate (2,3-BPG) sensitivity, which may result in heightened oxygen affinity. Notably, two variants have been previously documented at this location: the unstable Hb Atlanta and the high-affinity Hb Pasadena.

Optimization of antimicrobial peptides for the application against biocorrosive bacteria

Microbiologically influenced corrosion is a common problem in the industrial field due to the deterioration of metals in the presence of various microorganisms, in particular sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB). A common method to reduce microbiologically influenced corrosion is the application of biocides. The limited number of suitable biocides and the resulting development of resistance, high dosage, and high application rate hinder an effective application. An environmentally friendly alternative could be the application of antimicrobial peptides (AMP), which have already been established in the field of medical devices for a while. Here, the successful treatment of different AMPs against 3 SRB and 1 SOB was demonstrated. The peptide L5K5W was favored due to its broad activity, high stability, and simple structure resulting in low synthesis costs. An alanine scan showed that substitution of leucine with tryptophan increased the activity of this peptide twofold compared to the original peptide against D. vulgaris, the main representative of SRB. Additional optimization of this modified peptide through changes in amino acid composition and lipidations significantly increased the effectiveness, finally resulting in a minimum inhibitory concentration (MIC) of 15.63 μg/mL against Desulfovibrio vulgaris. Even against the marine SRB Desulfovibrio indonesiensis with a required salt concentration of min. 2%, an activity of the peptides can be observed (MIC: 31.25 μg/mL). The peptides also remained stable and active for 7 days in the supernatant of the bacterial culture.Key points• Antimicrobial peptides provide an alternative to combat biocorrosive bacteria.• Optimization of the peptide sequence leads to a significant increase in activity.• The investigated peptides exhibit high stability, both in the medium and in the bacterial supernatant.Graphical abstract

Molecular analyses of the C-terminal CRAF variants associated with cardiomyopathy reveal their opposing impacts on the active conformation of the kinase domain

The germline mutations in the C-terminus of CRAF kinase, particularly L603, and S612T/L613V, are associated with congenital heart disorders, for example, dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). The experimental data suggest that genetic alternation at position 603 impairs, while those at positions 612/613 enhance the CRAF kinase activity. However, the underlying mechanistic details by which these mutations increase or decrease kinase activity remain elusive. Therefore, we applied molecular dynamic simulation to investigate the impacts of these point mutations on the conformation of the CRAF kinase domain. The results revealed that the substitution of Leucine 603 for proline transits the kinase domain to a state that exhibits the molecular hallmarks of an inactive kinase, for example, a closed activation loop, ‘αC-helix out’ conformation and a distorted regulatory hydrophobic spine. However, two HCM-associated variants (S612T and L613V) show features of an active conformation, such as an open activation loop conformation, ‘αC-helix in’, the assembly of the hydrophobic spine, and more surface-exposed catalytic residues of phosphoryl transfer reaction. Overall, our study provides a mechanistic basis for the contradictory effects of the CRAF variants associated with HCM and DCM.

Target-site and non-target site resistance mechanisms are associated with iodosulfuron resistance in Lolium perenne L.

ABSTRACT Resistance to iodosulfuron, a herbicide targeting acetolactate synthase (ALS), was identified and characterised in a population of Lolium perenne L. from Canterbury, New Zealand. The resistant population was 264 times more resistant to iodosulfuron than a susceptible population, based on a ratio of the rate corresponding to a 50% reduction in resistant and susceptible plant survival (R/S LD50). The resistant population was also 14-times more resistant to nicosulfuron, another ALS-inhibitor of the same class. However, no resistance to imazapyr, an ALS-inhibitor from a different chemical class, was detected in the resistant population. Pre-treatment with an inhibitor of cytochrome P450 reduced the level of iodosulfuron resistance to 46-fold in the resistant population, suggesting enhanced herbicide metabolism, a non-target site mechanism, contributed to resistance of this herbicide. In addition, a proline to leucine substitution at codon 197 was detected, suggesting a target-site-based mechanism was also associated with resistance to iodosulfuron. This is the first report on the resistance mechanisms to ALS-inhibiting herbicides identified in Lolium perenne in New Zealand and highlights the importance of integrated weed management approaches to reduce the likelihood of ALS-inhibitor herbicide resistance becoming more widespread.

Kir7.1 disease mutant T153I within the inner pore affects K+ conduction.

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.

MO022: Digenic inheritance in Alport syndrome

Abstract BACKGROUND AND AIMS The approach to Alport syndrome is a difficult task due to the phenotypic variability of its symptomatology, incomplete penetrance and its different forms of inheritance [1]. It presents a high degree of underdiagnosis, both because of erratic diagnosis as well as the existence of undiagnosed patients [2]. This study shows a patient carrying two pathogenic variants in COL4A3 and COL4A4 genes, respectively. The interest of the case lies in the low reported frequency of this type of inheritance, of which there are still no prevalence studies, but which may help to better understand this entity, as well as aid future diagnoses [3, 4]. METHOD Our index case is a 55-year-old male (IV.1). His medical history dates back to childhood when the disease began with microhematuria, repeated urinary tract infections, proteinuria and a progressive decrease in glomerular filtration rate until he required hemodialysis at 23 years. He received a living-renal transplantation from his mother, restarting hemodialysis at 49 years; a second engraftment was carried out 3 years later that was working for 5 years until a clear cell renal cancer was diagnosed and transplantectomy was required. There was no evidence of hearing or ocular impairment. Throughout the patient's follow-up, the existence of other relatives on the paternal side with kidney disease became known, and with the suspicion of hereditary origin, a genogram of the family was constructed with five generations [Figure 1]. A genetic study was performed using a next-generation sequencing (NGS) panel (Sophia Genetics) covering the coding and splicing regions of 44 genes related to HRE (Table 1). Subsequently, the study was extended to other relatives. RESULTS Molecular analysis identified two probably pathogenic variants in our index case and other relative at the moment, both in heterozygosis, one in exon 48 of the COL4A3 gene: c.4421T > C, p.(Leu1474Pro). This is a missense-type change in which thymine is replaced by cytosine at position 4421 of the coding sequence and predicts the substitution of the amino acid leucine by proline at position 1474 of the protein, affecting two functional domains. This variant is described and reported in the databases as pathogenic. On the other hand, it presents a deletion, also in heterozygosis, of exon 9 of the COL4A4 gene. Both variants were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA), respectively. In this family, the variants co-segregate with the disease, although the analysis of other cases would be useful; furthermore, both variants co-segregate together, which indicates that the variants are in cis and both come from the same branch and neither has a de novo origin. CONCLUSION We have identified a case of digenic inheritance, two pathogenic variants in COL4A3 and COL4A4 genes respectively, thanks to the NGS techniques, of which very few cases have been described in the literature. Genetic analysis is the only way to confirm the diagnosis, often even to establish it after uncertain diagnoses; it is also the way to determine the mode of transmission and can often avoid the use of other invasive and not risk-free techniques such as skin or kidney biopsy. Although there is currently no curative treatment, early diagnosis is important to slow its progression, so that after the identification of a pathogenic variant, the family implications should be of special interest, to carry out adequate genetic counseling where family members at risk are informed and genetic study is offered, as well as the existing reproductive options for affected patients, such as preimplantation genetic testing or gamete donation. Otherwise, the offspring should be included in a program for early detection and monitoring of the disease.

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry.

Proteorhodopsin (PR) is a photoactive proton pump found in marine bacteria. There are two phenotypes of PR exhibiting an environmental adaptation to the ocean's depth which tunes their maximum absorption: blue‐absorbing proteorhodopsin (BPR) and green‐absorbing proteorhodopsin (GPR). This blue/green color‐shift is controlled by a glutamine to leucine substitution at position 105 which accounts for a 20 nm shift. Typically, spectral tuning in rhodopsins is rationalized by the external point charge model but the Q105L mutation is charge neutral. To study this tuning mechanism, we employed the hybrid QM/MM method with sampling from molecular dynamics. Our results reveal that the positive partial charge of glutamine near the C14−C15 bond of retinal shortens the effective conjugation length of the chromophore compared to the leucine residue. The derived mechanism can be applied to explain the color regulation in other retinal proteins and can serve as a guideline for rational design of spectral shifts.

Complete Shift in Glycosyl Donor Specificity in Mammalian, but not C. elegans β1,4‐GalT1 Y286L mutants, Enables the Synthesis of N,N‐Diacetyllactosamine

AbstractN,N‐diacetyllactosamine (LacdiNAc) is a motif present in various glycoconjugates of animal cell surfaces. The enzymatic synthesis of the LacdiNAc motif was previously achieved by using mutant variants of the human and bovine β1,4‐galactosyltransferase 1 (β1,4‐GalT1, EC 2.4.1.38). These enzymes bear a specific tyrosine to leucine substitution, which enables the transfer of GalNAc from UDP‐GalNAc. Herein, we present that the same tyrosine to leucine substitution in recombinant β1,4‐GalT1 orthologues of mouse, rat, or pig also resulted in the complete shift of the glycosyl donor specificity from UDP‐galactose to UDP‐GalNAc. In addition, we tested a previously undescribed β1,4‐GalT1 orthologue from C. elegans (CeGalT1). Given that this enzyme possesses an isoleucine residue in the wild‐type variant, this enzyme should prefer UDP‐GalNAc as a glycosyl donor substrate. Surprisingly, CeGalT1 was strictly specific for UDP‐Gal, but an isoleucine to tyrosine mutant variant of CeGalT1 widened the substrate range towards UDP‐GalNAc. In addition, the CeGalT1 mutant variant showed also significantly improved enzymatic activity towards UDP‐Gal. To further investigate the role of this tyrosine to leucine substitution of β1,4‐GalT1 and to evaluate the potential of this mutation for biotechnological applications, we generated Tyr286Leu B4galt1 mice using CRISPR/Cas9 gene editing and screened the milk of these mice for its ability to synthesize N,N‐diacetyllactosamine.