Biology subjects

Marek, A.

Publications and source records attributed to Marek, A..

3 recordsLinked to original sources

bioRxiv2025

Allosteric factors in the calcium/calmodulin-responsive kinase II hub domain determine selectivity of GHB ligands for CaMKIIa

The Ca2+/CaM-dependent protein kinase II alpha (CaMKII) is a highly important synaptic protein, which comprises a unique holoenzyme structure organized via the central hub domain. Recently, a distinct binding pocket in the CaMKII hub domain was identified for the endogenous neuromodulator {gamma}-hydroxybutyric acid (GHB) and related synthetic analogues. Key interacting residues in CaMKII were revealed, but the pronounced selectivity towards the alpha variant of CaMKII has remained unresolved. Aimed at elucidating the molecular determinants for this selectivity, we here conducted binding studies to CaMKII-HEK whole-cell homogenates using two different in-house-developed GHB-related radioligands, [3H]HOCPCA and [3H]O-5-HDC, in combination with site-directed mutagenesis. Binding to CaMKII with the smaller-type radioligand [3H]HOCPCA validated key involvement of the four known residues (His395, Arg433, Arg453 and Arg469), but also revealed a role for the upper hub flexible loop containing the CaMKII-specific residue Trp403 (Leu in all other CaMKII isozymes). Insertion of the corresponding residues (L467W/C533R) into CaMKII{beta} failed to introduce [3H]HOCPCA binding. However, with the larger-type radioligand, [3H]O-5-HDC, specific binding in CaMKII{beta} (L467W/C533R) was achieved. Thus, of the four native CaMKII isozymes, only CaMKII accommodates GHB ligands. The study identifies the CaMKII flexible pocket loop as a distantly located "allosteric" factor in determining selectivity of GHB analogues for CaMKII. It sheds light on a remarkable interplay of the entire hub cavity for accommodation of ligands, and corroborates GHB analogues as CaMKII-selective.

biochemistry↗

bioRxiv2023

The image-based ultrasonic cell shaking test

Mechanical signals play a vital role in cell biology and is a vast area of research. Thus, there is motivation to understand cell deformation and mechanobiological responses. However, the ability to controllably deform cells in the ultrasonic regime and test their response is a noted challenge throughout the literature. Quantifying and eliciting an appropriate stimulus has proven to be difficult, resulting in methods that are either too aggressive or oversimplified. Furthermore, the ability to gain a real-time insight into cell deformation and link this with the biological response is yet to be achieved. One application of this understanding is in ultrasonic surgical cutting, which is a promising alternative to traditional methods, but with little understanding of its effect on cells. Here we present the image based ultrasonic cell shaking test, a novel method that enables controllable loading of cells and quantification of their response to ultrasonic vibrations. Practically, this involves seeding cells on a substrate that resonates at ultrasonic frequencies and transfers the deformation to the cells. This is then incorporated into microscopic imaging techniques to obtain high-speed images of ultrasonic cell deformation that can be analysed using digital image correlation techniques. Cells can then be extracted after excitation to undergo analysis to understand the biological response to the deformation. This method could aid in understanding the effects of ultrasonic stimulation on cells and how activated mechanobiological pathways result in physical and biochemical responses.

bioengineering↗

bioRxiv2023

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.

biochemistry↗