Publications
2021
Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.
In recent decades, antibodies (Abs) have attracted the attention of academia and the biopharmaceutical industry due to their therapeutic properties and versatility in binding a vast spectrum of antigens. Different engineering strategies have been developed for optimizing Ab specificity, efficacy, affinity, stability and production, enabling systematic screening and analysis procedures for selecting lead candidates. This quality assessment is critical but usually demands time-consuming and labor-intensive purification procedures. Here, we harnessed the direct-mass spectrometry (direct-MS) approach, in which the analysis is carried out directly from the crude growth media, for the rapid, structural characterization of designed Abs. We demonstrate that properties such as stability, specificity and interactions with antigens can be defined, without the need for prior purification.
Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.
Understanding proteinligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)-based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct-MS method for rapid analysis of proteins directly from crude samples. Here, direct-MS enables label-free studies of proteindrug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including proteinprotein interactions, drug uptake and binding, and characterization of therapeutic proteins.
\u201cThe biggest challenge facing scientists is navigating in the unknown My favorite molecule is the proteasome complex \u201d Find out more about Michal Sharon in her Introducing Profile.
The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/ RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple βα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one β-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.
Accumulating evidence arising from numerous clinical studies indicate that assembled and functional 20S proteasome complexes circulate freely in plasma. Elevated levels of this core proteolytic complex have been found in the plasma of patients suffering from blood, skin and solid cancers, autoimmune disorders, trauma and sepsis. Moreover, in various diseases, there is a positive correlation between circulating 20S proteasome (c20S) levels and treatment efficacy and survival rates, suggesting the involvement of this under-studied c20S complex in pathophysiology. However, many aspects of this system remain enigmatic, as we still do not know the origin, biological role or mechanisms of extracellular transport and regulation of c20S proteasomes. In this review, we provide an overview of the current understanding of the c20S proteasome system and discuss the remaining gaps in knowledge.
Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.