DRAFT: This module has unpublished changes.

     Molecular cell biology 

     Another class that left a good lasting impression on me was molecular cell biology. In this class, we learned about the complex inner workings of the cell on a molecular level. We studied protein structures, DNA transcription and cell apoptosis. My scientific research papers are posted below. 



Henock Tsegaye
Prof. Bettica
Folding modification and degradation of protein structures
         From building the cell structure to fighting foreign pathogens to carrying chemical information across the body, proteins play an instrumental role in the vitality of every living organism. All protein molecules whether it is as simple as an insulin molecule or as complex as hemoglobin are made of the same monomers, the 20 basic amino acids, but the remarkable diversity and versatility of protein molecules come from not only the different combination with which the amino acids combine but also how they are arranged in space, known as protein folding. As important as proteins are in all cellular activities, it is therefore imperative that cells conduct various processes such as protein modification and protein degradation to successfully guide protein molecules to where they are needed, render them biologically active or even reduce them down to their building blocks.
             Proteins acquire their three dimensional shape through folding whereby a molecule composed of L-alpha amino acids interlinked by peptide bonds, a primary structure, succumbs to intermolecular forces and folds onto itself forming secondary, tertiary and quaternary structures. A closer look into secondary structures shows that they can either be alpha helices, beta pleated sheets or triple helices; conformations resulting from hydrogen bonding between a carbonyl oxygen atom of a peptide linkage and the hydrogen atom of an amino group (Stoker 2001). For instance, the triple stranded coiled-coil structure of collage represents its secondary structure conformation ( Ramachandran and kartha 1954). Ramachandran suggested that collagen is composed of three equivalent polypeptide chains, each wound into a left-handed helix with three residues per turn(1955). It was also found out that gly-pro-hypro was a common sequence in collagen whereby glycine residue occur every third position and three alpha helices are positioned at a 1100 around a common axis. This arrangement gives collagen its characteristic tensile strength. In more complex protein molecules, however, secondary structures play a different role.
             Secondary structures are folding intermediates that initiate and facilitate the formation of tertiary and quaternary structures(Wright et. al, 1998). A good example is the presence of a well-defined helical hairpin ( a motiff composed of a helix-turn-helix) between helices G and H of myoglobin, which makes it a folding initiation site (Matheson and Scheraga, 1978). As the side chains of the individual amino acids further interact with each other by forming disulfide bonds, salt bridges, hydrogen bonds and hydrophobic interactions, the protein assumes its tertiary structure(Stocker, 2001). Such types of disulfide linkages between sulfhydryl groups of two cystine amino acids can be observed in structures of Oxytocin and vasopressin. But perhaps, the highest level of protein organization is achieved in quaternary structures, in which multiple independent polypeptide chains each with its own tertiary conformation are held together with hydrophobic interactions. A classic example would be the oxygen carrying heterotrophic tetramer, hemoglobin. Its structural overview reveals two identical alpha chains bound with two identical b chains which are folded in such a way that they have ion-capture sites for iron atoms.
          Protein folding immediately follows synthesis of proteins, and within seconds the first secondary structures appear (Creighton, 1985; kim and Baldwin,1982). Since this process is rapid and chaotic, efficient and accurate folding is only guaranteed through the assistance of molecular chaperones, which serve to prevent protein misfolding and aggregation in the cell. As amino acid chains come off the ribosomal assembly line, chaperones such as trigger factor Hsp70 and Prefoldin stabilize elongating chains. Chaperones are basically long cylindrical complexes within which a newly formed protein chain may fold unimpaired by aggregation or neighboring chains (Hartl 2002).
         Once a protein molecule has been synthesized and folded into its respective shape, it is now ready to be transported to its correct cellular location-be it another organelle or extra cellular secretion. However, in the crowded environment of a cell, it is nearly impossible for a protein molecule to find its way without being modified. Modification is the addition of sugar molecules to core oligosaccharides that are already attached to the protein. The sugar complexes direct the protein to its final destination. For example, the journey of the hydrolyase enzyme to the organelle, endosome, starts with a modification of the enzyme’s oligosaccahride to form mannose-6 phosphate in the cis cisterna of the Golgi apparatus. The mannose -6 phosphate guides the protein to the trans citerna of the Golgi apparatus where Mannose-6 phosphate receptor protein awaits. Once bound to the M6P receptor, the protein is transported through a vesicle to the endosome. When it reaches the endosome, the hydrolyase enzyme is released after undergoing dephosphorylation to remove the 6-phosphate from the Mannose sugar(Grfiffths and simons 1986; Dahms et al., 1989). However, modification is not solely for the purpose of transport. Stocker suggests that in some cases modification can help in the assembly of complex molecules. To exemplify, modification through the addition of carbohydrate units to the 5-hydroxyl sine residues of collagen will assist in cross linking. They direct the assembly of collagen triple helices into more complex aggregations called collagen fibrils(2001).
           In the cell, regulation of proteins is highly crucial. And as important as protein synthesis is, protein degradation is necessary if the cell wants to digest and acquire amino acids from food particles, or convert precursor protein molecules into their final structures and so on. In Ubiquitin dependent protein degradation, proteins which were modified by the addition of the polypeptide Ubiquitin are degraded by 26S protease. This constant ubiquitination and de ubiquitination is important in cell regulatory mechanisms such as receptor mediated endocytosis (Hochstrasser, 1996) .Usually, the cleavage site consists of the amino acid, Proline.
           In conclusion, proteins are tools with which all cellular functions are executed. This remarkable feat is achieved through their ability to fold and conform to function specific shapes. In addition to that, proteins can modified and degraded for transport and cellular regulation, respectively.


Works cited

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“Structure and Function of Red Cell Surface Antigens-Daniels-2006-ISBT science Series.”
Wiley Online Library.web.18 Dec.2010.





Henock Tsegaye

Dr. Bettica

The use of Cholesterol sequestering drugs against HIV-1 internalization

              The human immunodeficiency virus (HIV) is the a viral agent responsible for causing a disease of the immune system known as Acquired immunodeficiency syndrome (AIDS), a pandemic disease which continues to claim the lives of many . And yet, no single cure could stop the rapid spread of this HIV virus. However, recent developments in the research of cholesterol sequestering drugs have showed promising results in impairing, even blocking the HIV-1 virus from entering the cell. Therefore, the depletion of cholesterol from the plasma membrane using non-toxic sequestering drugs such as cyclodextrins and filipin will likely result in the prevention of HIV-1 internalization.
              HIV is classified under the lentivirus of the Retroviridae family (Bernhard, 1960). Like all retroviruses, a single HIV virion consists of two strands of RNA molecule within its core that make up the genome of the virus. Also found in the core of the virus are three enzymes, namely reverse transcriptase, protease and integrase; enzymes used to assimilate the viral genome into the host DNA (Layne et al. 1992). Surrounding the core is the viral envelop, containing numerous glycoprotein complexes called spikes. Each spike is a trimer consisting of a Trans membrane glycoprotein(gp 41) with HR1 and HR2 peptide domains, and a surface glycoprotein (gp 120)(Schawaller et al. 1989; Weiss et al. 1990).
The process of HIV infection and cell entry involves the fusion of viral and cellular membranes. Initially, the surface glycoprotein gp 120 binds with the CD4 primary receptor and CXCR4/CCR5 co receptors located in cholesterol-rich lipid rafts on the surface of CD4+ human T cells, monocytes/macrophages and dendritic cells(Doms and Trono 2000). This interaction results in a conformational change of gp 120, exposing the underlying viral Trans membrane gp 41(Doms and Trono 2000). When gp 41 is released from gp120, the hydrophobic N-terminus of the gp 41 embeds itself into the cell membrane and the HR2 domain starts to coil around the HR1 domain(Moldovan et al. 2006). This process accomplishes two things. Firstly, it brings the virus into close proximity to the cell membrane and secondly, it destabilizes the adjacent cell membrane and propels the viral core into the cytoplasm.
              The most critical step in the prevention of the HIV-1 internalization is the CD4 receptor-gp120 glycoprotein binding. The reason being that the CD4 receptors and its co receptor CXCR4/CCR5 are primarily situated on lipid rafts. As Pike describes it, lipid rafts are regions of membranes with distinct, characteristic structural composition and appear to act as platforms to colocalize proteins in intracellular signaling pathways(2003). This nature of lipid rafts is directly attributed to their cholesterol and sphignolipid-rich composition(Simmons 2000). Enrichment of the otherwise fluid and chaotic membrane with cholesterol and shignolipids will allow close packing and add rigidity to the membrane, making it favorable for the attachment and anchoring of receptors such as CD4 and its co receptor(Brown and London 2000). The added rigidity of the phospholipid bilayer has to do with the molecular structure of cholesterol itself. As a steroid, its large cyclic rings plug the spaces between adjacent phopholipids, forcing them to pack tighter.
             HIV-1 replication critically depends on cholesterol(Campbell et al. 2002). And the stimulation of cholesterol efflux from macrophages significantly reduces infectivity of the virions (Guyader 2002). Cholesterol sequestering drugs like methyl b cyclodextrin function by forming chains of sugar polymers which turn into hydrophobic rings, creating an appealing environment for hydrophobic molecules like cholesterol. Treatment of CD4+ T human lymphocytes with methyl b cyclodextrin depletes plasma cholesterol and relocalizes raft-resident receptors into non-raft environment. As the lipid raft’s integrity is compromised, the conformational structure of CD4 receptors and its coreceptors will change and become inaccessible for gp120 binding(Doms 2000). Without the proper gp 120-CD4 binding, HIV-1 will not be able to efficiently internalize into the cytoplasm.
In conclusion, HIV-1 virion infiltrates CD4+ T human lymphocytes, dendritic cells, monocytes and macrophages by latching onto their primary surface receptors CD4 and its coreceptor CXCR4/CCR5, which are located on cholesterol and sphignolipid-rich lipid rafts. However, research has shown that depleting the plasma cholesterol using methyl b cyclodextrin will destabilize the lipid rafts and their receptors, significantly blocking the internalization of the HIV-1 virion.



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Doms, R.W. 2000. Beyond Receptor Expression the Influence of Receptor Conformation, Density, and Affinity in HIV-1 Infection. Virology 276:229-237.



DRAFT: This module has unpublished changes.