Biochemistry and Molecular Biology Compendium

Biochemistry and Molecular Biology Compendium

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Biochemistry and Molecular Biology Compendium

Binding-change model

Mechanism of ATP synthase. ATP is shown in red, ADP and phosphate in pink, and the rotating γ subunit in black.

Depiction of ATP synthase using the chemiosmotic proton gradient to power ATP synthesis through oxidative phosphorylation.

In the 1960s through the 1970s, Paul Boyer developed the binding change, or flip-flop, mechanism, which postulated that ATP synthesis is coupled with a conformational change in the ATP synthase generated by rotation of the gamma subunit. The research group of John E. Walker, then at the MRC Laboratory of Molecular Biology in Cambridge, crystallized the F1 catalytic-domain of ATP synthase. The structure, at the time the largest asymmetric protein structure known, indicated that Boyer's rotary-catalysis model was, in essence, correct. For elucidating this, Boyer and Walker shared half of the 1997 Nobel Prize in Chemistry. Jens Christian Skou received the other half of the Chemistry prize that year "for the first discovery of an ion-transporting enzyme, Na+

, K+

 -ATPase."

The crystal structure of the F1 showed alternating alpha and beta subunits (3 of each), arranged like segments of an orange around an asymmetrical gamma subunit. According to the current model of ATP synthesis (known as the alternating catalytic model), the proton-motive force across the inner mitochondrial membrane, generated by the electron transport chain, drives the passage of protons through the membrane via the FO region of ATP synthase. A portion of the FO (the ring of c-subunits) rotates as the protons pass through the membrane. The c-ring is tightly attached to the asymmetric central stalk (consisting primarily of the gamma subunit), which rotates within the alpha3beta3 of F1 causing the 3 catalytic nucleotide binding sites to go through a series of conformational changes that leads to ATP synthesis. The major F1 subunits are prevented from rotating in sympathy with the central stalk rotor by a peripheral stalk that joins the alpha3beta3 to the non-rotating portion of FO. The structure of the intact ATP synthase is currently known at low-resolution from electron cryo-microscopy (cryo-EM) studies of the complex. The cryo-EM model of ATP synthase suggests that the peripheral stalk is a flexible structure that wraps around the complex as it joins F1 to FO. Under the right conditions, the enzyme reaction can also be carried out in reverse, with ATP hydrolysis driving proton pumping across the membrane.

The binding change mechanism involves the active site of a β subunit's cycling between three states. In the "open" state, ADP and phosphate enter the active site; in the diagram to the right, this is shown in red. The protein then closes up around the molecules and binds them loosely — the "loose" state (shown in orange). The enzyme then undergoes another change in shape and forces these molecules together, with the active site in the resulting "tight" state (shown in pink) binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle of ATP production.

Signs and Symptoms of Ebola Virus Disease

Signs and Symptoms of Ebola Virus Disease

Symptoms may appear anywhere from 2 to 21 days after exposure to Ebola, but the average is 8 to 10 days.Ebola Zaire kills people quickly, typically 7 to 14 days after symptoms appear. A person can have the virus but not show any symptoms for as long as 3 weeks. People who survive can still have the virus in their system for weeks afterward. The virus has been detected in semen up to 7 weeks after recovery, according to the WHO. Humans are not infectious until they develop symptoms. A person infected with Ebola virus will typically develop a fever, headache, joint and muscle pain, a sore throat, and intense muscle weakness.

Signs and Symptoms of Ebola Virus Disease are as follows:

• High fever (usually higher than 38.3 °C (100.9 °F))
• Muscle and Joint aches
• Headache
• Sore throat  and Shortness of breath
• Chest pain and cough
• Red eyes
• Weakness
• Swelling
• Severe weight loss
• Chills
• Confusion
• Fatigue
• Nausea and Vomiting
• Diarrhea (may be bloody)
• Internal and External bleeding
• Bleeding, usually from the eyes
• Stomach Pain
• Hiccups
• Raised Rash
• Kidneys and Liver Failure

Recovery from Ebola depends on good supportive clinical care and the patient’s immune response. People who recover from Ebola infection develop antibodies that last for at least 10 years. Ebola virus disease is fatal in 50-90% of cases. The sooner a person is given care, the better the chances that they will survive.

Signs and Symptoms of Ebola Virus Disease

Replication of Ebola Virus

Replication of Ebola Virus

Ebola Virus do not replicate through any kind of cell division; rather, they use a combination of host and virally encoded enzymes, alongside host cell structures, to produce multiple copies of viruses. These then self-assemble into viral macromolecular structures in the host cell. The virus completes a set of steps when infecting each individual cell.

Following are the steps during the replication of Ebola Virus:

1. Attachment

First of all, there is attachment of virus to host receptors through GP glycoprotein which is endocytosed into vesicles in the host cell. Host DC-SIGN and DC-SIGNR play a role in virion attachment.

2. Viral Entry (Penetration)

The virion enters early endosomes by Macropinocytosis or clathrin-mediated endocytosis. 

A. Macropinocytosis

 In this process, ruffled segments of the host’s plasma membrane protrude outward from the cell and form invaginations where the virus utilizes glycoproteins in order to attach to the surface of the plasma membrane. Macropinocytosis is a process in which the Eukaryotic host cells form macropinosomes, segments of plasma membranes that extend out from the cell approximately 0.2-10 µm, in order to incorporate the virus into the cell. The formation of macropinosomes occurs spontaneously, as a result of the activation of various growth factors, or simultaneously with the intake of cellular molecules or extracellular fluid.

B. Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is the other means by which Ebolavirus enters the host cell. This process is very similar to macropinocytosis in that the plasma membrane forms invaginations that engulf the cell. However, clathrin-mediated endocytosis is different in that proteins on the surface of the host’s surface, and in particular clathrin, facilitate the attachment of the virus to the host’s cell surface. Glycoproteins are still used to attach the virus to the cell surface, and the NP-C1 cholesterol transporter still facilitates the fusion of the virus with endosomes and lysosomes and still allows the virus to escape into the cytoplasm. Without the NPC1 cholesterol transporter, Ebolavirus cannot leave the vesicle in order to replicate and cause infection in other cells.

To penetrate the cell, the viral membrane fuses with vesicle membrane, and the nucleocapsid is released into the cytoplasm.

In some culture cells, GP glycoprotein can be processed by host Cathepsin L andCathepsin B into 19kDa GP1. But this processing is not happening in all cells or for all ebolavirus. 19kDA GP1 interacts with host NPC1, which is highly expressed in dendritic cells.

Fusion of virus membrane with the vesicle membrane is triggered by either low pH orNPC1 binding.

3. Sequential Transcription

During transcription, the RNA genome is transcribed into seven monocistronic mRNAs whose length is determined by highly conserved start and stop signals.

The transcription process begins with the binding of the polymerase complex to a single binding site located within the leader region of the genome. The complex then slides along the RNA template and sequentially transcribes the individual genes in their 3’ to 5’ order. Encapsidated, negative-sense genomic ssRNA is used as a template for the synthesis (3′-5′) of polyadenylated, monocistronic mRNAs and, using the host cell’s ribosomes, tRNA molecules, etc., the mRNA is translated into individual viral proteins.

4. Replication

As viral protein levels rise, a switch occurs from translation to replication. Using the negative-sense genomic RNA as a template, a complementary +ssRNA is synthesized; this is then used as a template for the synthesis of new genomic (-)ssRNA, which is rapidly encapsidated

Replication presumably starts when enough nucleoprotein is present to encapsidate neo-synthetized antigenomes and genomes.

5. Budding

The newly formed nucleocapsids and envelope proteins associate at the host cell’s plasma membrane; budding occurs, destroying the cell.

These viruses recruit components of the cellular ESCRT (endosomal sorting complex required for transport) system to mediate host-assisted viral budding. SCRT complexes are normally used by the cell for biological functions involving membrane remodeling, such as intraluminal vesicle formation, autophagy or terminal stages of cytokinesis. The ESCRT family consists of ESCRT-0, ESCRT-I, ESCRT-II which are primarily involved in cargo sorting and membrane deformation, and ESCRT-III which cleaves the bud neck from its cytosolic face . In the last step, vps4 disassembles the complex. The budding reaction catalyzed by the ESCRT machinery has reversed topology when compared with most other budding processes in the cell, such as endocytosis and formation of transport vesicles.

6. Release

Finally, the virion is released.

Replication of Ebola Virus