Biology subjects

Vargas, E.

Publications and source records attributed to Vargas, E..

7 recordsLinked to original sources

bioRxiv2025

Domain-specific decoupling of co-chaperone and ligase functions in STUB1 underlies biochemical and clinical heterogeneity in SCA48

AO_SCPLOWBSTRACTC_SCPLOWThe carboxyl terminus of HSC70-interacting protein (CHIP, encoded by STUB1) integrates co-chaperone and E3 ubiquitin ligase activities to maintain proteostasis. Heterozygous STUB1 mutations cause the dominant cerebellar ataxia SCA48 through incompletely understood mechanisms. We characterized 13 SCA48-associated variants in the TPR and U-box domains using recombinant protein and cellular assays. TPR mutations retained intrinsic ligase activity but lost HSC70 binding, substrate ubiquitination efficiency, and protein stability. Conversely, U-box mutations abolished ligase function, induced aberrant high-molecular-weight oligomerization, and frequently elevated steady-state CHIP levels while only partially impairing co-chaperone activity. Many variants exhibited temperature-sensitive defects and stress-induced nuclear mislocalization. Principal component analysis revealed robust domain-specific biochemical clustering. RNA-seq following STUB1 knockdown demonstrated preserved HSF1-dependent transactivation but a loss of CHIP-dependent amplification of ubiquitination, chaperone, and transcriptional pathways under heat stress. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical profiles to upper motor neuron involvement and U-box profiles to prominent dysarthria. Collectively, SCA48 mutations decouple CHIPs dual functions in a domain-dependent manner, exerting dominant-negative or gain-of-toxic effects that drive the observed clinical heterogeneity. These findings establish a direct biochemical-clinical correlation in SCA48 and provide a framework for domain-targeted therapeutic strategies exploiting residual ligase or chaperone activity.

neuroscience↗

bioRxiv2025

Comprehensive 16S rRNA gene sequencing and meta-transcriptomic analyses in female reproductive tract microbiota: Two molecular profiles with different messages

In recent years, high-throughput sequencing technologies have revolutionised reproductive microbiome research. Our understanding of the endometrial microbiome is primarily based on DNA-based 16S rRNA gene profiling, but DNA detection does not imply alive microbe presence. While this method is cost-effective and widely used, it has notable limitations, including the underestimation of microbial diversity, abundance, and functionality, as well as limited species-level resolution. Meta-transcriptomic analysis, an RNA-based approach, addresses these shortcomings by capturing functional transcripts that are actively expressed in living microbes. This study aims to characterize the endometrial microbiota through a dual approach, by integrating 16S rRNA gene sequencing and meta-transcriptomic analyses. By simultaneously analysing microbial composition and gene expression within the female reproductive tract samples, we seek to provide a more comprehensive understanding of its microbiota and their functional potential. A total of 49 women, aged 27-42 years, were enrolled in the study. Vaginal swabs, endometrial brushings, and endometrial biopsy samples were collected from each participant during the mid-secretory menstrual cycle phase, 6-9 days post luteinizing hormone surge. Our findings suggest that in low-microbial-biomass environments like the endometrium, the correlation between 16S rRNA gene sequencing and meta-transcriptomics is relatively weak. This highlights the limitations of microbial analysis of low-microbial-biomass samples. Alternatively, it suggests that microbial functions and genome activity are tissue-specific and dependent on the host tissue environment. Moreover, RNA-based analysis provides higher resolution in detecting certain pathogens, even within the endometrium. In conclusion, our study underscores the need to integrate genomic and transcriptomic data in microbiota studies to better elucidate critical and health-related microbiota-host interactions.

genomics↗

bioRxiv2024

A PDZ-kinase allosteric relay mediates Par complex regulator exchange

The Par complex polarizes the plasma membrane of diverse animal cells using the catalytic activity of atypical Protein Kinase C (aPKC) to pattern substrates. Two upstream regulators of the Par complex, Cdc42 and Par-3, bind separately to the complex to influence its activity in different ways. Each regulator binds a distinct member of the complex, Cdc42 to Par-6 and Par-3 to aPKC, making it unclear how they influence one anothers binding. Here we report the discovery that Par-3 binding to aPKC is regulated by aPKC autoinhibition and link this regulation to Cdc42 and Par-3 exchange. The Par-6 PDZ domain activates aPKC binding to Par-3 via a novel interaction with the aPKC kinase domain. Cdc42 and Par-3 have opposite effects on the Par-6 PDZ-aPKC kinase interaction: while the Par-6 kinase domain interaction competes with Cdc42 binding to the complex, Par-3 binding is enhanced by the interaction. The differential effect of Par-3 and Cdc42 on the Par-6 PDZ interaction with the aPKC kinase domain forms an allosteric relay that connects their binding sites and is responsible for the negative cooperativity that underlies Par complex polarization and activity.

biochemistry↗

bioRxiv2024

Fiber-based Probes for Electrophysiology, Photometry, Optical and Electrical Stimulation, Drug Delivery, and Fast-Scan Cyclic Voltammetry In Vivo

Recording and modulation of neuronal activity enables the study of brain function in health and disease. While translational neuroscience relies on electrical recording and modulation techniques, mechanistic studies in rodent models leverage genetic precision of optical methods, such as optogenetics and imaging of fluorescent indicators. In addition to electrical signal transduction, neurons produce and receive diverse chemical signals which motivate tools to probe and modulate neurochemistry. Although the past decade has delivered a wealth of technologies for electrophysiology, optogenetics, chemical sensing, and optical recording, combining these modalities within a single platform remains challenging. This work leverages materials selection and convergence fiber drawing to permit neural recording, electrical stimulation, optogenetics, fiber photometry, drug and gene delivery, and voltammetric recording of neurotransmitters within individual fibers. Composed of polymers and non-magnetic carbon-based conductors, these fibers are compatible with magnetic resonance imaging, enabling concurrent stimulation and whole-brain monitoring. Their utility is demonstrated in studies of the mesolimbic reward pathway by simultaneously interfacing with the ventral tegmental area and nucleus accumbens in mice and characterizing the neurophysiological effects of a stimulant drug. This study highlights the potential of these fibers to probe electrical, optical, and chemical signaling across multiple brain regions in both mechanistic and translational studies.

neuroscience↗

bioRxiv2022

Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3

The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Through a biochemical reconstitution approach utilizing purified, intact Par complex and qualitative binding assays, we found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. We show that the interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.

cell biology↗

bioRxiv2021

Energetic determinants of the Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (Par-6 and atypical Protein Kinase C-aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it has been unclear how each contributes to the overall interaction. Using purified, intact Par complex and a quantitative binding assay, we found that energy for this interaction is provided by Par-3s second and third PDZ protein interaction domains. Both Par-3 PDZ domains bind to aPKCs PDZ Binding Motif (PBM) in the Par complex, with binding energy contributed from aPKCs adjacent catalytic domain. In addition to highlighting the role of Par-3 PDZ interactions with the aPKC kinase domain and PBM in stabilizing Par-3 - Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization.

cell biology↗

bioRxiv2019

Spherical Spindle Shape Promotes Perpendicular Cortical Orientation by Preventing Isometric Cortical Pulling on both Spindle Poles during C. elegans Female Meiosis

Meiotic spindles are positioned perpendicular to the oocyte cortex to facilitate segregation of chromosomes into a large egg and a tiny polar body. In C. elegans, spindles are initially ellipsoid and parallel to the cortex before shortening to a spherical shape and rotating to the perpendicular orientation by dynein-driven cortical pulling. The mechanistic connection between spindle shape and rotation has remained elusive. Here we used mutants of the microtubule-severing protein katanin to manipulate spindle shape without eliminating cortical pulling. In a katanin mutant, spindles remained ellipsoid, had pointed poles and became trapped in either a diagonal or a parallel orientation. Results indicated that astral microtubules emanating from both spindle poles initially engage in cortical pulling until microtubules emanating from one pole detach from the cortex allowing pivoting of the spindle. The lower viscous drag experienced by spherical spindles prevented recapture of the cortex by astral microtubules emanating from the detached pole. In addition, maximizing contact between pole dynein and cortical dynein stabilizes round poles in a perpendicular orientation. Spherical spindle shape can thus promote perpendicular orientation by two distinct mechanisms.

cell biology↗