Biology subjects

Trzeciakiewicz, H.

Publications and source records attributed to Trzeciakiewicz, H..

4 recordsLinked to original sources

bioRxiv2025

Synergistic MAPT mutations as a platform to uncover modifiers of tau pathogenesis

The natively unfolded tau (MAPT) protein is extremely soluble, which poses challenges when modeling neurofibrillary tangle (NFT) pathology in Alzheimers disease (AD). To overcome this hurdle, we combined P301L and S320F mutations (PL-SF) to generate a rapid and reliable tau pathology platform to expedite the discovery of factors that modify tau aggregation. Using this model, we evaluated heat-shock proteins (Hsp), which have been linked to tau pathology, but whose role in AD remains enigmatic and controversial. In primary neurons, expression of Hsp70, but not Hsc70 or Hsp90, exacerbated tau aggregation. Conversely, lowering Hsp70 or employing a chaperone-deficient tau mutant (PL-SF-4{Delta}) reduced tau phosphorylation and abrogated tau aggregation, highlighting Hsp70 as a key driver of tau aggregation. Hsp70 foci clustered within and surrounding neuritic plaques and NFTs in post-mortem AD brain. Functionally, mature aggregate-bearing neurons showed deficits in neuronal firing and network communication, which were restored by chaperone-binding deficient tau variants that abrogated tau pathology. This study provides a powerful cell-intrinsic model for accelerated tau aggregation, which can be harnessed to identify potent regulators of tau aggregation as promising therapeutic targets.

neuroscience↗

bioRxiv2023

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and -synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and -synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

bioengineering↗

bioRxiv2023

Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates

1.Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimers and Parkinsons diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimers disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both transgenic tau mouse models and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

bioengineering↗

bioRxiv2021

RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control

The brain expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntingtons disease where it promotes the clearance of mutant Huntingtin protein. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.

neuroscience↗