Biology subjects

Boshnakovska, A.

Publications and source records attributed to Boshnakovska, A..

3 recordsLinked to original sources

bioRxiv2024

The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this proteins functions gives new insights into the role of BAP31 in development and pathology.

cell biology↗

bioRxiv2024

Profiling the LAM family of contact site tethers provides insights into their regulation and function

Membrane contact sites are molecular bridges between organelles that are sustained by tethering proteins and enable organelle communication. The endoplasmic reticulum (ER) membrane harbors many distinct families of tether proteins that enable the formation of contacts with all other organelles. One such example is the LAM (Lipid transfer protein At Membrane contact sites) family, composed of six members, each containing a lipid binding and transfer domain and an ER-embedded transmembrane segment. The family is divided into three homologous pairs each unique in their molecular architecture and localization to different ER subdomains. However, what determines the distinct localization of the different LAMs and which specific roles they carry out in each contact are still open questions. To address these, we utilized a labeling approach to profile the proximal protein landscape of the entire family. Focusing on unique interactors we could support that Lam5 resides at the ER-mitochondria contact site and demonstrate a role for it in sustaining mitochondrial activity. Capturing shared interactors of multiple LAMs, we show how the Lam1/3 and Lam2/4 paralogous pairs could be associated specifically with the plasma membrane. Overall, our work provides new insights into the regulation and function of the LAM family members. More globally it demonstrates how proximity labeling can help identify the shared or unique functions of paralogous proteins.

cell biology↗

bioRxiv2024

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Delta/+ mice hearts

BackgroundPhospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14{Delta}-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14{Delta}-PLN. MethodsWe developed an improved complexome profiling (CP) workflow specifically geared towards identifying disruption of very high molecular-weight (>2 MDa) protein complexes and supercomplexes in presymptomatic R14{Delta}/+ mice hearts. Ventricular tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) proteomics analysis. Systematic analysis of CP data using conventional strategies yielded limited insights, likely due to underrepresentation of cardiac-specific complexes in the curated protein complex databases used as ground-truth for analysis. We thus developed PERCOM: a novel data analysis strategy that does not rely upon protein complex databases and can, furthermore, be implemented on widely available spreadsheet software. ResultsSEC-DIA-MS coupled with PERCOM identified 296 proteins with disrupted elution profiles in presymptomatic 28wk-old R14{Delta}/+ mice. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Alterations to mitochondrial and ICD supercomplexes were observed in mice as young as 9wks of age and were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. ConclusionUsing a novel CP workflow, we identify mitochondrial alterations as an early-stage R14{Delta}-PLN event and provide preliminary data showing effects at the ICD. These molecular components underlie critical cardiac functions and their alteration at a young age may contribute to R14{Delta}-PLN pathogenesis.

cell biology↗