Unassembled cell wall proteins form aggregates in the extracellular space of Chlamydomonas reinhardtii strain UVM4.

The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.

Profiling Carbonylated Proteins in Heart and Skeletal Muscle Mitochondria from Trained and Untrained Mice.

Understanding the relationship between physical exercise, reactive oxygen species, and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Heart and skeletal muscles have a high density of mitochondria with robust energetic demands, and mitochondria plasticity has an important role in both the cardiovascular system and skeletal muscle responses. The aim of this study was to investigate the influence of regular physical activity on the oxidation profiles of mitochondrial proteins from heart and tibialis anterior muscles. To this end, we used the mouse as animal model. Mice were divided into two groups: untrained and regularly trained. The carbonylated protein pattern was studied by two-dimensional gel electrophoresis followed by Western blot with anti-dinitrophenyl hydrazone antibodies. Mass spectrometry analysis allowed the identification of several different protein oxidation sites, including methionine, cysteine, proline, and leucine residues. A large number of oxidized proteins were found in both untrained and trained animals. Moreover, mitochondria from skeletal muscles and heart showed almost the same carbonylation pattern. Interestingly, exercise training seems to increase the carbonylation level mainly of mitochondrial proteins from skeletal muscle.

Ferritin nanocage-enabled detection of pathological tau in living human retinal cells.

Tauopathies, including Alzheimer's disease and Frontotemporal Dementia, are debilitating neurodegenerative disorders marked by cognitive decline. Despite extensive research, achieving effective treatments and significant symptom management remains challenging. Accurate diagnosis is crucial for developing effective therapeutic strategies, with hyperphosphorylated protein units and tau oligomers serving as reliable biomarkers for these conditions. This study introduces a novel approach using nanotechnology to enhance the diagnostic process for tauopathies. We developed humanized ferritin nanocages, a novel nanoscale delivery system, designed to encapsulate and transport a tau-specific fluorophore, BT1, into human retinal cells for detecting neurofibrillary tangles in retinal tissue, a key marker of tauopathies. The delivery of BT1 into living cells was successfully achieved through these nanocages, demonstrating efficient encapsulation and delivery into retinal cells derived from human induced pluripotent stem cells. Our experiments confirmed the colocalization of BT1 with pathological forms of tau in living retinal cells, highlighting the method's potential in identifying tauopathies. Using ferritin nanocages for BT1 delivery represents a significant contribution to nanobiotechnology, particularly in neurodegenerative disease diagnostics. This method offers a promising tool for the early detection of tau tangles in retinal tissue, with significant implications for improving the diagnosis and management of tauopathies. This study exemplifies the integration of nanotechnology with biomedical science, expanding the frontiers of nanomedicine and diagnostic techniques.

Combined Delivery of miR-15/16 through Humanized Ferritin Nanocages for the Treatment of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a widespread type of leukemia that predominantly targets B lymphocytes, undermining the balance between cell proliferation and apoptosis. In healthy B cells, miR-15/16, a tandem of microRNAs, functions as a tumor suppressor, curbing the expression of the antiapoptotic B cell lymphoma 2 protein (Bcl-2). Conversely, in CLL patients, a recurring deletion on chromosome 13q14, home to the miR15-a and miR16-1 genes, results in Bcl-2 overexpression, thereby fostering the onset of the pathology. In the present research, a novel approach utilizing humanized ferritin-based nanoparticles was employed to successfully deliver miR15-a and miR-16-1 into MEG01 cells, a model characterized by the classic CLL deletion and overexpression of the human ferritin receptor (TfR1). The loaded miR15-a and miR16-1, housed within modified HumAfFt, were efficiently internalized via the MEG01 cells and properly directed into the cytoplasm. Impressively, the concurrent application of miR15-a and miR16-1 demonstrated a robust capacity to induce apoptosis through the reduction in Bcl-2 expression levels. This technology, employing RNA-loaded ferritin nanoparticles, hints at promising directions in the battle against CLL, bridging the substantial gap left by traditional transfection agents and indicating a pathway that may offer hope for more effective treatments.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae.

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.