Month: November 2021

Mitochondrial VDAC1: A Key Gatekeeper as Potential Therapeutic Target

Mitochondria are the key source of ATP that fuels cellular functions, and they are also central in cellular signaling, cell division and apoptosis. Dysfunction of mitochondria has been implicated in a wide range of diseases, including neurodegenerative and cardiac diseases, and various types of cancer. One of the key proteins that regulate mitochondrial function is the voltage-dependent anion channel 1 (VDAC1), the most abundant protein on the outer membrane of mitochondria. VDAC1 is the gatekeeper for the passages of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis due to its interaction with apoptotic and anti-apoptotic proteins, namely members of the Bcl-2 family of proteins and hexokinase. Therefore, regulation of VDAC1 is crucial not only for metabolic functions of mitochondria, but also for cell survival. In fact, multiple lines of evidence have confirmed the involvement of VDAC1 in several diseases. Consequently, modulation or dysregulation of VDAC1 function can potentially attenuate or exacerbate pathophysiological conditions. Understanding the role of VDAC1 in health and disease could lead to selective protection of cells in different tissues and diverse diseases. The purpose of this review is to discuss the role of VDAC1 in the pathogenesis of diseases and as a potentially effective target for therapeutic management of various pathologies.

The VDACs are the most abundant protein in the OMM with a molecular weight of approximately 32 kD. Under normal physiological conditions, VDACs function in tandem with the IMM adenine nucleotide translocase (ANT), via the mitochondrial creatine kinase (mCK) in the IMS. The conformational states of VDAC are voltage-dependent and exhibit different selectivity and permeability for small ions, showing a preference for anions in the open state and for cations in the closed state. Therefore, they are considered the principal sites for the exchange of metabolites and small solutes between the IMS and the cytosol. The efficient transfer of energy metabolites across mitochondria depends on the interaction between VDAC, mCK, and ANT, ANT/mCK/VDAC, which is fostered by physiological [Ca2+].

Single Chain Variable Fragment (scFv) Molecules

Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. 

The conventional antibody consists of two heavy chains and two light chains connected with disulfide bonds. The antibody structure can be divided into a constant Fc domain (crystallizable fragment domain) and the Fab fragment (antibody binding fragment) contains the Fv domains (variable fragment domains) at the end of both the arms. In humans, the antibody synthesized is usually glycosylated in the Fc region, which stabilizes the antibody and is necessary for the antibody-dependent immune responses. Enzymatic cleavage of antibody at the N-terminal side of the inter-heavy chain disulfide bridges results in the formation of Fc and Fab fragments. There are two variable regions in a Fab fragment interact with the antigen and each of these units represent the smallest functional antigen-binding domain.

The scFv can be generated by amplifying the variable regions of the Fab fragment from the mAb and by linking it together with a flexible peptide linker (usually (GGGGS)3). Advances in molecular techniques further improved the prospect of engineering scFv to improve its specificity, avidity, affinity, and half-life. Multimerization of the variable domains using, the linker, the tetramerization domain of a native protein like p53, the leucine zippers ,or the C-terminal fragment of C4-binding protein improved the affinity of the scFv to a great extent. The immunogenicity generated by the Fc portion of the antibody is absent in the conventional scFv molecule. The scFv expressing together with the Fc region of IgG (scFv-Fc) is found to be beneficiary with its “effector” functions in many reports. Antibody fragments of therapeutic and diagnostic importance have been expressed in mammalian systems plants and in prokaryotes. An extensive review of the biotechnological applications of antibody fragments has been given earlier. Plant expressed antibodies are also used in studying the basic metabolism of plants in terms of disease resistance against a pathogen, protein–protein interactions or the specific role of an endogenous protein in a metabolic process by selectively modulating its activity.

Advantages of Antibody Fragments and Biopharming

Expression of mAbs in heterologous production systems is precarious as the biological activity of the resultant molecule is dependent on several post-translational modifications. Biosynthesis of conventional antibody molecules (150 kDa), through the mammalian expression system and transgenic animals is highly expensive and time-consuming. The scFvs are smaller in size (~ 30 kDa) with less post-translational modifications. They show a similar specificity and affinity of the parental antibody against the antigen. Due to the smaller size, scFvs show a rapid blood clearance (a useful property in the radiotherapy and other diagnostic applications) and better tissue penetration (which has greater impact when they are used as therapeutics) than the full length mAbs. Due to the rapid blood clearance, the in vivo availability of scFv is low compared to the mAbs, which is considered as the major drawback of scFv. In order to increase the bioavailability of scFv in the bloodstream, the diabodies (~ 55 k Da), triabodies (~ 85 kDa) and tetrabodies (~ 120 kDa) were developed by manipulating the length of the linker peptide in between the VH and VL domains or by using the methods which facilitate the multimerization of scFv molecules. Tandem dimerization of the scFv generated from two different antibody sources can also create a bispecific antibody by the variation in the arrangement of the VH and VL sequences and was proved to be an effective methods to detect specific cell types with fluorescence-tagged scFv dimers. Further to that, the multivalency generated by combining the coding sequences of more than one scFv together with the multimerization is used to link different moieties (one may be targeting a cell surface receptor on a cancer cell and other bound to an anti-cancer drug) which facilitate the targeted delivery of the therapeutics to its site of action.