Biology subjects

Knight, A.

Publications and source records attributed to Knight, A..

8 recordsLinked to original sources

bioRxiv2024

Molecular determinants of protein pathogenicity at the single-aggregate level

Determining the structure-function relationships of protein aggregates is a fundamental challenge in biology. These aggregates, whether formed in vitro, within cells, or in living organisms, present significant heterogeneity in their molecular features such as size, structure, and composition, making it difficult to determine how their structure influences their functions. Interpreting how these molecular features translate into functional roles is crucial for understanding cellular homeostasis and the pathogenesis of various debilitating diseases like Alzheimers and Parkinsons. In this study, we introduce a bottom-up approach to explore how variations in protein aggregates size, composition, post-translational modifications and point mutations profoundly influence their biological functions. Applying this method to Alzheimers and Parkinsons associated proteins, we uncover the mechanism of novel disease-relevant pathways and demonstrate how subtle alterations in composition and morphology can shift the balance between healthy and pathological states. Our findings establish a broadly applicable framework for investigating protein dysfunctions in various proteinopathies.

biochemistry↗

bioRxiv2024

Undruggable oncoproteins cMyc and NMyc bind to mediator of transcription with superior high affinity

The overexpression of MYC genes is frequently found in many human cancers including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. The nine-amino-acid activation domain (9aaTAD) has been identified using our prediction algorithm in all four Yamanaka factors including c-Myc and showed to activate transcription as short peptides. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription. We discovered strong interactions in nanomolar scale between the 9aaTAD of c-Myc and N-Myc proteins with the KIX domain of CBP coactivator. The c-Myc 9aaTAD (region 98-108) was not overlapping with the MBII (region 128-143) and therefore represents TRRAP independent activation region. Next, we showed the 9aaTADs in human c-Myc and N-Myc conservation within the MYC family. Interestingly, the loss of the 9aaTAD in L-Myc paralogs was identified in higher metazoans suggesting the deletions had occurred in early tetrapod evolution. In summary, as c-Myc is largely intrinsically disordered protein and therefore difficult to target by small molecule inhibitors, our finding of the c-Myc 9aaTAD in complex with the KIX domain represents a promising druggable target for development of new peptide inhibitors in MYC-driven tumors.

biochemistry↗

bioRxiv2023

Spontaneous transfer of small peripheral peptides between supported lipid bilayer and giant unilamellar vesicles

Vesicular trafficking facilitates material transport between membrane-bound organelles. Membrane protein cargos are trafficked for relocation, recycling, and degradation during various physiological processes. In vitro fusion studies utilized synthetic lipid membranes to study the molecular mechanisms of vesicular trafficking and to develop synthetic materials mimicking the biological membrane trafficking. Various fusogenic conditions which can induce vesicular fusion have been used to establish synthetic systems that can mimic biological systems. Despite these efforts, the mechanisms underlying vesicular trafficking of membrane proteins remain limited and robust in vitro methods that can construct synthetic trafficking systems for membrane proteins between large membranes (>1 m2) are unavailable. Here, we provide data to show the spontaneous transfer of small membrane-bound peptides ([~]4 kD) between a supported lipid bilayer (SLB) and giant unilamellar vesicles (GUVs). We found that the contact between the SLB and GUVs led to the occasional but notable transfer of membrane-bound peptides in a physiological saline buffer condition (pH 7.4, 150 mM NaCl). Quantitative and dynamic time-lapse analyses suggested that the observed exchange occurred through the formation of hemi-fusion stalks between the SLB and GUVs. Larger protein cargos with a size of [~]77 kD could not be transferred between the SLB and GUVs, suggesting that the larger-sized cargos limited diffusion across the hemi-fusion stalk, which was predicted to have a highly curved structure. Our system serves as an example synthetic platform that enables the investigation of small-peptide trafficking between synthetic membranes and reveals hemi-fused lipid bridge formation as a mechanism of peptide transfer. Graphical abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/544237v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f72d8corg.highwire.dtl.DTLVardef@cf7ed5org.highwire.dtl.DTLVardef@8fee0forg.highwire.dtl.DTLVardef@1565484_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Evaluating Sleep Disturbances in Children with Rare Genetic Neurodevelopmental Syndromes

BackgroundAdequate sleep is important for proper neurodevelopment and positive health outcomes. Sleep disturbances are more prevalent in children with genetically determined neurodevelopmental syndromes compared to typically developing counterparts. We characterize sleep behavior in Rett (RTT), Angelman (AS) and Prader-Willi (PWS) syndromes in order to identify effective approaches for treating sleep problems in these populations. We compared sleep-related symptoms across individuals with these different syndromes to each other, and to typically developing controls. MethodsChildren were recruited from the Rare Diseases Clinical Research Network (RDCRN) consortium registries; unaffected siblings were enrolled as related controls. For each participant, a parent completed multiple sleep questionnaires including: Pediatric Sleep Questionnaire (Sleep-Disordered Breathing [SDB]); Childrens Sleep Habits Questionnaire; Pediatric Daytime Sleepiness Scale. ResultsSleep data were analyzed from 714 participants, ages 2-18 years. Young children with AS had more reported sleep problems than children with RTT or PWS. Older children with RTT had more reported daytime sleepiness than those with AS or PWS. Finally, all individuals with RTT had more evidence of sleep-disordered breathing when compared to individuals with PWS. Notably, typically developing siblings were also reported to have sleep problems, except for sleep-related breathing disturbances which were associated with each of the genetic syndromes. ConclusionsIndividuals with RTT, AS and PWS frequently experience sleep problems, including sleep-disordered breathing. Screening for sleep problems in individuals with these and other neurogenetic disorders should be included in clinical assessment and managements. These data may also be useful in developing treatment strategies and in clinical trials.

genetics↗

bioRxiv2019

The 9aaTAD activation domains in the four Yamanaka Oct4, Sox2, Myc, and Klf4 transcription factors essential during the stem cell development

Somatic cells can be reprogrammed by the Yamanaka factors Oct4, Sox2, Myc and Klf4 activators into induced pluripotent stem cells. Throughout their genome, the Oct4, Sox2 and Klf4 cooperate with mediators of transcription, where the DNA binding sites serve as scaffolds for the phase-separated transcriptional condensates at distinct genome loci. In this study, we identified the 9aaTAD activation domains as the common interaction interface of the Yamanaka factors for transcription machinery. All four activation domains were identified by our online 9aaTAD prediction service and experimentally confirmed as strong activators of transcription. We considered the mediator interactions granted by 9aaTADs as part of the Yamanaka factors ability to reprogram cell fate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/876706v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@c5105forg.highwire.dtl.DTLVardef@1b5e910org.highwire.dtl.DTLVardef@1d83c2aorg.highwire.dtl.DTLVardef@1561881_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs

The Sp1 transcription factor has been defined as glutamine-rich activator. The Nine amino acid TransActivation Domains (9aaTAD) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in the Sp1 and in all nine members of SP family with broad natural 9aaTAD variations. We showed by the amino acid substitutions that the glutamine residues are completely dispensable for 9aaTADs function. We described the 9aaTAD domains origin and evolutionary history. The ancestral Sp2 gene with inactive 9aaTAD has duplicated in early chordates and created new paralogs Sp1, Sp3 and Sp4. We discovered that the accumulation of valines in the 9aaTADs correlated with the domain inactivation. The Sp2 activation domain, whose dormancy have lasted over 100 million years during chordate evolution, enabled later diversification in the Sp1-4 clade, including both repressors and activators. The new paralogs Sp1 and Sp3 activation domains have regained their original activator function by loss of valines in their 9aaTADs.

molecular biology↗

bioRxiv2018

How to overcome the ATRA resistance with the 9aaTAD activation domains in retinoic acid receptors

In higher metazoa, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor cofactors NCoA, which are surprisingly absent in lower metazoa. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor activates transcription as a small peptide. We showed, that the 9aaTAD domains are conserved in the human nuclear hormone receptors including HNF4, RARa, VDR and PPARg. The small 9aaTAD peptides derived from these nuclear hormone receptors also effectively activated transcription and that in absence of the NCoA adaptors. We identified adjacent inhibitory domains in the human HNF4 and RARa, which hindered their activation function.\n\nIn acute promyelocytic leukaemia (PML-RARa), the receptor mutations often caused all-trans retinoic acid (ATRA) resistance. The fact that almost the entire receptor is needed for ATRA mediated receptor activation, this activation pathway is highly susceptible for loss of function when mutated. Nevertheless in the most of the reported mutants, the activation domains 9aaTAD are still intact. The release of activation 9aaTAD from its dormancy by a new drug might be the sound strategy in combat the ATRA resistance in PML leukaemia.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC=\"FIGDIR/small/298646_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (36K):\norg.highwire.dtl.DTLVardef@187575borg.highwire.dtl.DTLVardef@782db0org.highwire.dtl.DTLVardef@1ff9fe7org.highwire.dtl.DTLVardef@15b27ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2017

Gal4 activation domain 9aaTAD could be inactivated by adjacent mini-inhibitory domain and reactivated by distal re-activation domain

The characterisation of the activation domains started three decades ago with Gcn4 and Gal4 activators. The amorphous character of the activation domains strongly hindered their definition. Moreover, during the attempts to localise the Gal4 activation domain, the artificial peptides, an unintended consequence of cloning, were responsible for artificial transcriptional activity of the several Gal4 constructs. These artefacts produced enormous experimental bias and misconception. The presence of inhibitory domains in some Gal4 constructs made the misperception even worse. Previously, we reported that the nine amino acid transactivation domain, 9aaTAD, is the exclusive activation domain in the Gal4 protein. The activation domain 9aaTAD could be identified in Gal4 paralogs Oaf1, Pip2, Pdr1, Pdr3 and other activators p53, E2A and MLL. Surprisingly, the activation domain 9aaTAD was reported as misconception for Gal4 activator. Here we demonstrated that small region of 10 amino acids adjacent to the Gal4 activation domain 9aaTAD is an inhibitory domain, which the authors included in their constructs. Moreover, we identified Gal4 region, which was able to the reverse the inhibitory effect. The 9aaTAD re-activation domain was localized to the 13 amino acid long region. In this report we clarified the numerous confusions and rebutted supposed 9aaTAD misconception.\n\nSummaryThe activation domain 9aaTAD has decisive function in Gal4 activation. Gal4 activation domain 9aaTAD could be inhibited by adjacent region of 10 amino acids. The inhibited Gal4 activation domain 9aaTAD could be reactivated by 13 amino acid long Gal4 region. The activation domains 9aaTAD could be identified by our 9aaTAD prediction algorithm, especially in the Gal4 family.

molecular biology↗