The Establishment of Cell Biology

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18/05/20 Biology Reference this

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Dr Günter Blobel was awarded a Nobel prize in 1999 in the field of medicine and physiology for discovering that proteins encode intrinsic signals that allow for their transportation and localization in cells (Nobel Media AB, 2019). His contribution towards protein topogenesis has since been a very critical step in understanding diseases for improving human health and in the advancement of the biotechnology field. This report aims to inform the reader on the history of cell biology before the contributions of Dr Blobel, the journey towards uncovering the critical finding that is the signal hypothesis and also to show more recent studies stemming from the work of the awardee. 

 

Research in cell biology prior to the contributions of Dr Blobel had already demonstrated findings showing the fluidity of membranes (Gorter & Grendel, 1925), their role as diffusion barriers for macromolecules, and the fact that transfer of macromolecules is critical to physiological function (Singer & Nicolson, 1972; Skalova et al., 2017). Furthermore, previous advancements of electron microscopy and fractionation methods allowing separation of organelles meant scientists could visualize the pathway of secretory proteins in cells from synthesis to excursion which won Claude, de Duve and Palade a Nobel prize (Nobel Media AB, 2019a). Around the time of Palade and his colleague’s discoveries, Dr Blobel gained interest in understanding how thousands of different proteins reach their respective destinations in cells (Nobel Media AB, 2019b). 

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The signalling hypothesis was first postulated by Blobel and Sabatini in 1971 (figure 1) to show a possible way in which ribosome-membrane interactions may occur (Blobel & Sabatini, 1971). The critical point to this idea was that proteins encode zip codes that determine the translocation of proteins to the endoplasmic reticular (ER) membrane (Blobel & Sabatini, 1971). The postulated signal hypothesis model originated due to an initial investigation of possible compositional differences between ER-bound ribosomes and free ribosomes through deconstruction experiments using high concentrations of salt and puromycin to release nascent polypeptides from ribosomes to give defined components, which were used to rebuild a cell-free system that recapitulates protein translation and translocation across microsomal membrane vesicles (Blobel & Sabatini, 1971a).

Figure 1. The first version of the signal hypothesis proposed by Blobel and Sabatini (1971) shows a continuous cycle of ribosome assembly and detachment to mRNA and assumes all mRNAs translated on bound ribosomes have common features in the codons around the 5’ N-terminus end (X) which are recognized by a binding factor to mediate binding to the ER-membrane. The cycle ends when the ribosome dissociates and releases the polypeptide chain into the intracisternal space (Blobel & Sabatini, 1971).

Blobel and Dobberstein hypothesis at the time was highly refuted but they managed to provide evidence a year later by reproducing experiments from Swan et al, (1972) and Milstein et al, (1972) by disrupting ribosome-microsome complexes undergoing translation in an in-vitro translation system then allowing for detached ribosomes to continue translation in the absence of microsomes (Milstein, Brownlee, Harrison, & Mathews, 1972; Swan, Aviv, & Leder, 1972). Outputs from SDS-PAGE and autoradiography showed that the small mature protein appeared before the precursor chain and also that the more extended the period after protein-membrane disruption with ATA the larger the produced product (Blobel & Dobberstein, 1975). Furthermore, readout experiments Blobel and Dobberstein (1975) did using mild proteolysis demonstrated that mature secretory proteins are localized in microsomes which protect them from degradation (Blobel & Dobberstein, 1975), support by later work on rough microsomes from dog pancreas showed proteolytic enzymes did not degrade the light chain, and in the absence of microsomes only the large light chain is produced but gets degrades when exposed to proteolytic enzymes (Blobel & Dobberstein, 1975a). 

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Blobel and Dobberstein also showed that the presence of immunoglobulin was not an artefact of translation in a heterologous system as detached ribosomes isolated from the detergent treatment of rough microsomes had both nascent light chains that were unprocessed  and nascent chains that were already proteolytically processed in-vivo (Blobel & Dobberstein, 1975). It was also shown that a signal sequence is cleaved during translation and a binding factor that is incorporated in the microsomal membrane acts as a translocation channel that has selectivity for nascent chains encoding the signal sequence which allows for interaction of the membrane with the ribosome and that only mRNA with translation products to be secreted contained this amino acid signal codon immediately right of the initiation codon (Blobel & Dobberstein, 1975; Meyer, 1982). In 1984, the final signal hypothesis (see figure 2 for steps) was revised to overall show that as early entry stages of secretory protein to the ER take place, the nascent polypeptide chain enters the membrane channel due to presence of a translocation channel consisting of a signal recognition particle (SRP) made of protein and mRNA and its associated membrane spanning SRP-receptor (Walter & Blobel, 1982) which mediates the transport of the signal containing chain through direct binding (Meyer, 1982; Simon & Blobel, 1991). The model also shows presence of a signal peptidase allowing for endoproteolytic removal of the signal sequence in the ER-lumen (Blobel and Dobberstein 1975; Jackson & Blobel, 1977).

Figure 2. The last diagrammatically revised signal hypothesis by Walter, Gilmore & Blobel (1984) revealing the recycling cycle of the SRP and SRP-receptor after correct targeting of the ribosome to the membrane (a-e) and the ribosome cycle (1-7). The free (a), ribosome-bound (b) and membrane-bound (e) forms of the SRP are present in equilibrium. Initially, as the translation of the coding sequence containing mRNA occurs (1-3), there is an increase in affinity of binding between the SRP and the signal sequence which causes the arrest of elongation on the initiated polypeptide chain just after the emergence of the signal sequence from the ribosome. Then, The SRP-arrested ribosome interacts with the SRP receptor on the ER-membrane to release arrest of elongation (4) and the SRP (d). Once targeting to the membrane is fulfilled, the ribosome interacts with other integral membrane proteins to form a functional ribosome junctions complex (4-6). A pore-like channel forms to allow for translocation of the nascent polypeptide in extended form into the ER-membrane (5). Enzymatic interactions occur in the lumen involving signal peptidase, which cleaves the signal sequence. Finally (6,7) The complete polypeptide is released in the ER-lumen, and the ribosome detaches to enter the soluble pool so that the ribosome cycle repeats (Adapted from Protein Translocation across the Endoplasmic Reticulum, by P. Walter, R. Gilmore, & G. Blobel, 1984, Cell, 38 (1), p. 5-8).

The initial and subsequent knowledge gained by the work of  Blobel and his colleagues regarding protein translation and translocation has been essential, since around 25% of proteins are likely to cross a membrane in order to reach their assigned destination (Dalbey, Kuhn, & Berliner, 2019) and moreover, has made an immense contribution towards scientific understanding of many medically important processes like the topogenic signalling in our immune system to produce antibodies (Nobel Media AB, 2019). Since a majority of current drug targets are integral proteins, it has been possible to create new specific drugs that get targeted to particular organelles (Dalbey, Kuhn, & Berliner, 2019). The signal hypothesis, allowed for our understanding of diseases caused by transport defects such as that of cystic fibrosis which has a loss of signal peptidase consequently resulting in abnormal physiochemical properties of proteins observed in the disease (Garg et al., 2015). Applications of the signal hypothesis  looking at prion proteins demonstrated that these proteins typically get directed to the outer leaflet of membranes once passed across the ER (Miesbauer et al., 2010). In yeast, the putative transmembrane domain is what leads to consequences of protein mislocalization due to the incorrect folding or misprocessed of prion proteins causing them to interfere with cell viability when they bind onto the membrane (Heller et al., 2003). In mammals, prion misfolding is linked to causing neurotoxic effects contributing to neurodegeneration in Alzheimer disease (Heller et al., 2003). The mechanism of targeting prion proteins to the ER involves steps from signal hypothesis described by Blobel, such as the presence of an N-terminal signal sequence and recognition of both the signal sequence and the ribosome by SRP to result in the cease of elongation and targeting of the complex to sec61 translocon, but when SRP binds to form a complex with its SRP-receptor at the ER-membrane synthesis is allowed to proceed. Since the Nobel award, research in identification at atomic resolution of a wide range of molecular functions in transport including ATPase activity using methods such as Real-time imaging, NMR, X-ray crystallography and cryo-EM techniques (Dalbey, Kuhn, & Berliner, 2019).

In conclusion, the dedication by Dr Blobel and efforts of collaborating scientists to continue experimentation and refinement of the signal hypothesis model  despite rejections has paved the way into redefining the cell biology field. The signal hypothesis demonstrated great importance and foundation to our understanding of cell transport mechanisms to date and is an effective, although not the only means of describing translocations in cells. Knowledge can be transferred to the many fields of science such as microbiology, chemistry and medicine to help in the understanding of mechanisms underlying diseases to allow production of new useful drugs for application in cell and gene therapies. Overall, without a doubt, Blobel is a highly deserving awardee of the Nobel prize. 

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