1, Describe the ultrastructure and function of the Endoplasmic Reticulum

The Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a series of interconnected membranous sacs and tubules
that collectively modifies proteins and synthesizes lipids. However, these two functions are
performed in separate areas of the ER: the rough ER and the smooth ER. The hollow portion
of the ER tubules is called the lumen or cisternal space. The membrane of the ER, which is a
phospholipid bilayer embedded with proteins, is continuous with the nuclear envelope.

Rough ER
The rough endoplasmic reticulum (RER) is so named because the ribosomes attached to its
cytoplasmic surface give it a studded appearance when viewed through an electron
microscope . Ribosomes transfer their newly synthesized proteins into the lumen of the RER
where they undergo structural modifications, such as folding or the acquisition of side chains.
These modified proteins will be incorporated into cellular membranes—the membrane of the
ER or those of other organelles—or secreted from the cell (such as protein hormones,
enzymes). The RER also makes phospholipids for cellular membranes. If the phospholipids
or modified proteins are not destined to stay in the RER, they will reach their destinations via
transport vesicles that bud from the RER's membrane. Since the RER is engaged in
modifying proteins (such as enzymes, for example) that will be secreted from the cell, the
RER is abundant in cells that secrete proteins. This is the case with cells of the liver, for
example.

Smooth
ER
The smooth endoplasmic reticulum (SER) is continuous with the RER but has few or no
ribosomes on its cytoplasmic surface. Functions of the SER include synthesis of
carbohydrates, lipids, and steroid hormones; detoxification of medications and poisons; and
storage of calcium ions. In muscle cells, a specialized SER called the sarcoplasmic reticulum
is responsible for storage of the calcium ions that are needed to trigger the coordinated
contractions of the muscle cells.
2. Describe the role of DNA and RNA in protein synthesis:
Proses sintesis protein berlangsung dalam dua tahap yaitu transkripsi dan translasi. Proses
tersebut berlangsung di organel sel yakni ribosom dan sitoplasma. Adapun urutan proses
dalam sintesis protein secara ringkasnya sebagai berikut:
1. DNA melakukan transkripsi (mencetak RNA-d) untuk membawa kode-kode
membentuk protein berdasarkan pada urutan basa nitrogennya.
2. RNA-d melepaskan diri dari DNA dan membawa kode-kode genetik (kodon) keluar
dari nukleus menuju ke ribosom di dalam sitoplasma. RNA-d bertindak sebagai
cetakan (matriks). Di ribosom ini RNA-d melekat pada RNA ribosom (RNA-r).
3. RNA-t yang ada di dalam sitoplasma datang dengan membawa asam amino yang
selesai dengan kode-kode yang dibawa oleh RNA-d. RNA-t ini melekat
(berpasangan) dengan RNA-d sesuai dengan pasangan-pasangan basa nitrogennya
(dengan tripel RNA-t).
4. Asam-asam amino yang dibawa oleh RNA-t akan saling bergandengan dan
membentuk rangkaian rantai polipeptida sampai terbentuk protein yang diharapkan di
dalam ribosom.
Transkripsi merupakan proses pembentukan RNA-m dari DNA yang terjadi di dalam
nukleus. RNA-m inilah yang nantinya berperan sebagai pembawa pesan dari DNA menuju
ribosom agar informasinya dapat diolah menjadi protein. Pada proses translasi, sintesis
polpeptida dengan urutan spesifik berdasarkan rantai mRNA yang dibuat pada tahapan
transkripsi. Susunan asam amino yang dibentuk sesuai dengan susunan basa nitrogen dalam
kodon. Satu per satu asam amino berikatan dengan triplet anticodon (tRNA) sehingga
membentuk polipeptida. Sehingga dapat disimpulkan bahwa apabila tidak ada DNA dan
RNA maka sintesis protein tidak akan berjalan.

3. Amino acids are biologically important organic compounds containing amine (-NH2) and
carboxyl (-COOH)functional groups, along with a side-chain (R group) specific to each
amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen,
though other elements are found in the side-chains of certain amino acids. About 500 amino
acids are known (though only 20 appear in the genetic code) and can be classified in many
ways. They can be classified according to the core structural functional groups' locations as
alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to
polarity, pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing
hydroxyl or sulfur, etc.). In the form of proteins, amino acids comprise the second-largest
component (water is the largest) of human muscles, cells and other tissues. Outside proteins,
amino acids perform critical roles in processes such as neurotransmitter transport and
biosynthesis.
As both the amine and carboxylic acid groups of amino acids can react to form amide
bonds, one amino acid molecule can react with another and become joined through an amide
linkage. This polymerization of amino acids is what creates proteins. This condensation
reaction yields the newly formed peptide bond and a molecule of water. In cells, this reaction
does not occur directly; instead, the amino acid is first activated by attachment to a transfer
RNA molecule through an ester bond. This aminoacyl-tRNA is produced in an ATP-
dependent reaction carried out by an aminoacyl tRNA synthetase.This aminoacyl-tRNA is
then a substrate for the ribosome, which catalyzes the attack of the amino group of the
elongating protein chain on the ester bond. As a result of this mechanism, all proteins made
by ribosomes are synthesized starting at their N-terminus and moving toward their C-
terminus.
However, not all peptide bonds are formed in this way. In a few cases, peptides are
synthesized by specific enzymes. For example, the tripeptideglutathione is an essential part of
the defenses of cells against oxidative stress. This peptide is synthesized in two steps from
free amino acids. In the first step, gamma-glutamylcysteine synthetase condenses cysteine
and glutamic acid through a peptide bond formed between the side-chain carboxyl of the
glutamate (the gamma carbon of this side-chain) and the amino group of the cysteine. This
dipeptide is then condensed with glycine by glutathione synthetase to form glutathione.
In chemistry, peptides are synthesized by a variety of reactions. One of the most-used
in solid-phase peptide synthesis uses the aromatic oxime derivatives of amino acids as
activated units. These are added in sequence onto the growing peptide chain, which is
attached to a solid resin support. The ability to easily synthesize vast numbers of different
peptides by varying the types and order of amino acids (using combinatorial chemistry) has
made peptide synthesis particularly important in creating libraries of peptides for use in drug
discovery through high-throughput screening.

4. Describe the structural forms of polypeptides and how th sequencing of amino acids is
determined
The Peptide Bond
If the amine and carboxylic acid functional groups in amino acids join together to form amide
bonds, a chain of amino acid units, called a peptide, is formed. A simple tetrapeptide
structure is shown in the following diagram. By convention, the amino acid component
retaining a free amine group is drawn at the left end (the N-terminus) of the peptide chain,
and the amino acid retaining a free carboxylic acid is drawn on the right (the C-terminus). As
expected, the free amine and carboxylic acid functions on a peptide chain form a zwitterionic
structure at their isoelectric pH.
By clicking the "Grow Peptide" button, an animation showing the assembly of this peptide
will be displayed. The "Show Structure" button displays some bond angles and lengths that
are characteristic of these compounds.

Primary Structure
There are 20 different standard L-α-amino acids used by cells for protein construction.
Amino acids, as their name indicates, contain both a basic amino group and an acidic
carboxyl group. This difunctionality allows the individual amino acids to join together in long
chains by forming peptide bonds: amide bonds between the -NH2 of one amino acid and the -
COOH of another. Sequences with fewer than 50 amino acids are generally referred to as
peptides, while the terms protein or polypeptideare used for longer sequences. A protein can
be made up of one or more polypeptide molecules. The end of the peptide or protein
sequence with a free carboxyl group is called the carboxy-terminus or C-terminus. The terms
amino-terminus or N-terminus describe the end of the sequence with a free α-amino group.

Secondary Structure
Stretches or strands of proteins or peptides have distinct characteristic local structural
conformations or secondary structure, dependent on hydrogen bonding. The two main types
of secondary structure are the α-helix and the ß-sheet.

The α-helix is a right-handed coiled strand. The side-chain substituents of the amino acid
groups in an α-helix extend to the outside. Hydrogen bonds form between the oxygen of the
C=O of each peptide bond in the strand and the hydrogen of the N-H group of the peptide
bond four amino acids below it in the helix. The hydrogen bonds make this structure
especially stable. The side-chain substituents of the amino acids fit in beside the N-H groups.

The hydrogen bonding in a ß-sheet is between strands (inter-strand) rather than within strands
(intra-strand). The sheet conformation consists of pairs of strands lying side-by-side. The
carbonyl oxygens in one strand hydrogen bond with the amino hydrogens of the adjacent
strand. The two strands can be either parallel or anti-parallel depending on whether the strand
directions (N-terminus to C-terminus) are the same or opposite. The anti-parallel ß-sheet is
more stable due to the more well-aligned hydrogen bonds.

Tertiary Structure
The overall three-dimensional shape of an entire protein molecule is the tertiary structure.
The protein molecule will bend and twist in such a way as to achieve maximum stability or
lowest energy state. Although the three-dimensional shape of a protein may seem irregular
and random, it is fashioned by many stabilizing forces due to bonding interactions between
the side-chain groups of the amino acids.

The formation of disulfide bridges by oxidation of the sulfhydryl groups on cysteine is an

important aspect of the stabilization of protein tertiary structure, allowing different parts of
the protein chain to be held together covalently. Additionally, hydrogen bonds may form
between different side-chain groups. As with disulfide bridges, these hydrogen bonds can
bring together two parts of a chain that are some distance away in terms of sequence. Salt
bridges, ionic interactions between positively and negatively charged sites on amino acid side
chains, also help to stabilize the tertiary structure of a protein.

Quaternary Structure
Many proteins are made up of multiple polypeptide chains, often referred to as protein
subunits. These subunits may be the same (as in a homodimer) or different (as in a
heterodimer). The quaternary structure refers to how these protein subunits interact with
each other and arrange themselves to form a larger aggregate protein complex. The final
shape of the protein complex is once again stabilized by various interactions, including
hydrogen-bonding, disulfide-bridges and salt bridges.
5. Describe the normal process of protein synthesis

First, Transcription : Transcription is the process of RNA synthesis, controlled by the

interaction of promoters and enhancers. Several different types of RNA are produced,

including messenger RNA(mRNA), which specifies the sequence of amino acids in the

protein product, plustransfer RNA (tRNA) and ribosomal RNA (rRNA), which play a role

in the translation process.

Transcription involves three steps:

1. Initiation. The DNA molecule unwinds and separates to form a small open complex.
RNA polymerase binds to the promoter of the template strand.

2. Elongation. RNA polymerase moves along the template strand, synthesising an mRNA
molecule. In prokaryotes RNA polymerase is a holoenzyme consisting of a number of
subunits, including a sigma factor (transcription factor) that recognises the promoter. In
eukaryotes there are three RNA polymerases: I, II and III. The process includes a
proofreading mechanism.

3. Termination. In prokaryotes there are two ways in which transcription is terminated.

InRho-dependent termination, a protein factor called "Rho" is responsible for disrupting the
complex involving the template strand, RNA polymerase and RNA molecule. In Rho-
independent termination, a loop forms at the end of the RNA molecule, causing it to detach
itself. Termination in eukaryotes is more complicated, involving the addition of additional
adenine nucleotides at the 3' of the RNA transcript (a process referred to aspolyadenylation).

Translation : In translation the mature mRNA molecule is used as a template to assemble a

series of amino acids to produce a polypeptide with a specific amino acid sequence. The
complex in the cytoplasm at which this occurs is called a ribosome. Ribosomes are a mixture
of ribosomal proteins and ribosomal RNA (rRNA), and consist of a large subunit and a small
subunit.
Translation involves four steps:

1. Initiation. The small subunit of the ribosome binds at the 5' end of the mRNA
molecule and moves in a 3' direction until it meets a start codon (AUG). It then forms a
complex with the large unit of the ribosome complex and an initiation tRNA molecule.

2. Elongation. Subsequent codons on the mRNA molecule determine which tRNA

molecule linked to an amino acid binds to the mRNA. An enzyme peptidyl transferase links
the amino acids together using peptide bonds. The process continues, producing a chain of
amino acids as the ribosome moves along the mRNA molecule.

3. Termination. Translation in terminated when the ribosomal complex reached one or

more stop codons (UAA, UAG, UGA). The ribosomal complex in eukaryotes is larger and
more complicated than in prokaryotes. In addition, the processes of transcription and
translation are divided in eukaryotes between the nucleus (transcription) and the cytoplasm
(translation), which provides more opportunities for the regulation of gene expression.

6. Discuss the biological functions of proteins.

Proteins play a central role in biological processes. For example, proteins catalyse
reactions in our bodies, transport molecules such as oxygen, keep us healthy as part of the
immune system and transmit messages from cell to cell.
Some examples of proteins in our body are:
· Enzymes are proteins that facilitate biochemical reactions, for example, pepsin is a
digestive enzyme in your stomach that helps to break down proteins in food.
· Antibodies are proteins produced by the immune system to help remove foreign
substances and fight infections.
· DNA-associated proteins regulate chromosome structure during cell division and/or
play a role in regulating gene expression, for example, histones andcohesin proteins.

● Contractile proteins are involved in muscle contraction and movement, for example,
actin and myosin.
● Structural proteins provide support in our bodies, for example, the proteins in our
connective tissues, such as collagen and elastin.
● Hormone proteins co-ordinate bodily functions, for example, insulin controls our
blood sugar concentration by regulating the uptake of glucose into cells.
● Transport proteins move molecules around our bodies, for example,haemoglobin
transports oxygen through the blood.
7. Describe enzymes and their roles in protein synthesis

A. Enzyme : proteins that participate in cellular metabolic processes with the ability to
enhance the rate of reaction between biomolecules.

Enzyme acts as catalyst because of their three dimensional protein structure. This structure is
particularly sensitive to changes in pH, salts, and temperatures. Extremely high temperature
can irreversibly alter both the 3D structure of the enzyme and its activity.

Enzyme lowers the activation energy required in a reaction.

Enzymes are classified into :

· Oxidoreductase : catalyze redox reactions

· Transferase : transfer a group from one molecule to another

· Hydrolase : cleave bonds by adding water

· Lyase : catalyze removal of groups to form double bond, or the reverse

· Isomerase : catalyze intramolecular rearrangements

· Ligase : bond two molecules together

Major types of enzyme : protease, amylase, lipase, and cellulase. These enzymes break down
: protein, carbohydrate, fat, and cellulose respectively.

B. Protein synthesis mainly involves two processes, the transcription and translation.

During the process, the role of certain enzymes become crucial as described below :

· In transcription, there is an enzyme called RNA polymerase that unwinds the DNA
double strand and synthesizes primary transcript of RNA from DNA template.

· In translation, an enzyme called aminoacyl tRNA synthetase (aaRS) serves to charge

the tRNA by attaching the appropriate amino acid onto it. The tRNA then delivers the amino
acid to the ribosome based on the codon sequence of mRNA attached to the ribosome.

8. Describe the abnormalities which can occur in protein synthesis.

● Creutzfeldt-Jakob disease (CJD) is a rare, degenerative, invariably fatal brain
disorder. It affects about one person in every one million people per year worldwide;
in the United States there are about 300 cases per year. CJD usually appears in later
 life and runs a rapid course. Typically, onset of symptoms occurs about age 60, and
about 90 percent of individuals die within 1 year. In the early stages of disease, people
may have failing memory, behavioral changes, lack of coordination and visual
disturbances. As the illness progresses, mental deterioration becomes pronounced and
involuntary movements, blindness, weakness of extremities, and coma may occur.
● BSE (bovine spongiform encephalopathy) is a progressive neurological disorder of
cattle that results from infection by an unusual transmissible agent called a prion. The
nature of the transmissible agent is not well understood. Currently, the most accepted
theory is that the agent is a modified form of a normal protein known as prion protein.
For reasons that are not yet understood, the normal prion protein changes into a
pathogenic (harmful) form that then damages the central nervous system of cattle.

9. Define and describe the origin and role of prions and how abnormal prions cause disease

spongiform encephalopathies (TSEs) are a family of diseases of humans and animals

characterized by spongy degeneration of the brain with severe and fatal neurological signs
and symptoms.
In animals, scrapie is a common disease in sheep and goats. Mink and North American mule
deer and elk can contract TSEs. Bovine spongiform encephalopathy (BSE) is also a TSE,
affecting a number of species (cattle, human, cats, some types of animals in 300 settings).
BSE is a transmissible, neuro-degenerative fatal brain disease of cattle. The disease has a
long incubation period of 4-5 years and it is fatal for cattle within weeks to months of its
onset. The nature of the BSE agent is still being debated.
Strong evidence currently available supports the theory that the agent is composed largely, if
not entirely, of a self-replicating protein, referred to as a prion. It is transmitted through the
consumption of BSE-contaminated meat and bone meal supplements in cattle feed.

10. Review the pathophysiology of subacute spogioform

Variant Creutzfeldt-Jakob Disease and Bovine Spongiform Encephalopathy

Pathophysiology
Convincing evidence indicates that variant Creutzfeldt-Jakob disease (CJD) is a new disease.
Despite its name, variant CJD appears to be a human variant of BSE derived from a cow-to-
human species switch, rather than an actual variant of human sporadic CJD. BSE and variant
CJD are similar on the basis of patterns of infectable mouse strains, incubation time, survival
time, lesion distribution in the mouse brain, PrPSc gel banding patterns, and neuropathology,
which are readily distinguishable from other TSEs, such as scrapie and sporadic CJD.
Pathological investigation shows characteristic spongiform change and gliosis in the brain.
These changes are most predominant in basal ganglia and cerebellum.
Because no occupational exposure of patients with variant CJD to cattle on farms or in
abattoirs has been identified, spread is likely to occur through consumption of BSE-
contaminated meat products. Whether PrPSc (prion protein, scrapie isoform) can be
demonstrated in skeletal muscles remains controversial. However, a high-sensitivity Western
blotting technique identified muscle PrPSc in 8 of the 17 patients studied, although in much
lower concentrations than in the cerebral cortex, suggesting a potential role for skeletal
muscle in the transmission of variant CJD. Despite this evidence, the infection probably
resulted from beef products contaminated by nervous tissue, because neural tissues have a
much higher concentration of PrPSc than other peripheral tissues.
The amount of infectious agent ingested and host susceptibility, as determined by the human
genotype at PRNP codon 129, appear to play important roles in the development of variant
CJD. However, how oral consumption of BSE-contaminated beef leads to infection of the
CNS is unknown. In the early preclinical stages of the disease, PrPSc can be detected in
lymphoid tissues, suggesting a possible route of transmission from the gut. Prions probably
cross the mucosa via transmembranous tunneling of the membranous epithelial cells (M-
cells) and come in contact with the mucosa-associated lymphoid system, including Peyer
patches, where accumulation is found first.
A functional immune system is required for prion replication and transport outside of the
CNS. Mechanisms of further prion transport to other compartments of the lymphoreticular
system (LRS) are unclear. Prions accumulate in cells of the LRS, most prominently in the
follicular dendritic cells and in sympathetic nerve endings in the LRS. Then, prions reach the
CNS via splanchnic nerves at the level of the thoracic spinal cord and via parasympathetic
fibers connecting with the brain. The other possible route is blood, which was suggested by
experiments showing BSE transmission from sheep to sheep by blood transfusion.
Variant CJD is known to affect the brain, lymphoreticular system, pituitary and adrenal
glands, and gastrointestinal tract. One case report identified protease-resistant prion protein in
the dura mater, liver, pancreas, kidney, ovary, uterus, and skin of a patient with variant CJD,
indicating that organ involvement may actually be even more widespread.

Variant CJD in the blood supply

Concern is widespread that the blood supply might be contaminated with the variant CJD
agent. This possibility is supported by evidence that BSE in sheep can be transmitted by
blood transfusion. This concern progressed to fear with a report of a patient who died of
variant CJD 6.5 years after receiving a transfusion of red blood cells donated by an individual
who subsequently developed variant CJD. The authors do not present direct evidence that the
disease was transmitted by blood transfusion, but the chance that this case is not transfusion
related is very small. One more case of blood transfusion–related variant CJD has been
reported. This case is unique and very important in terms of its implications. This patient died
from a non-neurologic disorder 5 years after receiving a blood transfusion from a donor who
subsequently developed variant CJD, and he had no symptoms suggestive of variant CJD at
the time of his death. Protease-resistant prion protein (PrPres) was detected in the spleen and
a cervical lymph node but not in the brain. He was heterozygote at codon 129 of PRNP,
suggesting that susceptibility to variant CJD infection is not confined to methionine
homozygous PRNP genotype. In the late 1990s, the United Kingdom implemented strategies
for risk reduction. These included importation of plasma from the United States for
preparation of plasma derivatives (i.e., clotting factors), disposal of certain surgical
instruments, and universal leukoreduction in transfusions. There have been a total of four
affected individuals from the United Kingdom who received non-leucoreduced red cell
concentrates from UK donors. This possibility, combined with the probable existence of
subclinical carriers, raises the specter of an iatrogenic human-to-human wave of variant CJD
transmission. This again highlights the need for reliable detection methods for prion-tainted
blood products. In addition, a finding of preclinical infection in a patient heterozygous at
codon 129 of PRNP has significant implications regarding the future estimates and
surveillance of variant CJD.
5)describe the normal of Sintesis protein
Secara pasti dalam rantai RNA ini akan tersusun di dalam inti atau mRNA dan mRNA itu
akan keluar menuju lubang kecil atau pori-pori nuklir dari nukleus. Pergerakan ini akan
menuju pada area yang lebih besar dalam suatu sel yang bersangkutan atau di sitoplasma.
Dengan kemudian akan keluar dari inti dan mRNA ini secara terstruktur akan berfungsi
sebagai tempat berhentinya sintesis protein. Pada titik inilah terdapat kehadiran dari satu sub
unit ribosom.

Selama mRNA mengikat sub unit ribosom ini akan memicu adanya pendekatan lain melalui
rantai RNA yang disebut dengan RNA transfer. Deretan tRNA ini akan mencapai pada suatu
tempat yang tepat untuk mengikat mRNA dan menempel. Sembari menyentuh asam amino
pada salah satu di bagian ujungnya.

Saat hal ini terjadi, sub unit lain dari sel ribosom akan kembali dan membentuk struktur
lengkap. Ketikan ribosom ini menyelimuti helai dari RNA maka akan ada untaian lain dari
tRNA yang mendekat. Sehingga untaian ini akan membawa asam amino lain dan berbeda
dari yang sebelumnya. Peristiwa ini pun terjadi pada lokasi yang tepat agar dapat mengikat
mRNA dengan baik.

Saat helai dari kedua tRNA ini terjadi di asam amino maka akan ada dua asam amino yang
saling berikatan satu sama lain. Proses pengikatan ini akan terbantu oleh ribosom dan energi
dari sel lain untuk membentuk adenosin trifosfat atau ATP.

Proses ini berurutan dang saling berulang-ulang hingga rantai asam amino tumbuh lagi dan
lagi. Bilamana asam amino ini secara keseluruhan telah berada di urutan yang benar maka
rantai akan dilipatkan ke dalam bentuk tiga dimensi yakni ketika protein telah selesai
diproduksi.

Pada proses terciptanya protein dari dua sub unit ribosom ini terpisah nantinya akan
bergabung lagi untuk digunakan dalam waktu nanti. Tak luput dari proses sintesis protein
yang berlangsung di ribosom dan di seluruh sel. Proses ini berlangsung secara efisien dan
mensintesis ratusan protein dalam hitungan per detiknya.

Peran RNA dalam sintesis protein

Terdapat peran dari RNA yang begitu teramat penting dari proses sintesis protein ini. Sebab
messeger RNA ini akan menyediakan ribosom cetak biru untuk menciptakan protein.

Kemudian diimbangi pula dengan asam amino sebagai bahan pembangunan protein. Peran
pokok RNA disini ialah melawan infeksi yang dapat menghambat proses pembentukan
protein. Dengan begitu dapat meminimalisir adanya resiko dari ketidakfungsian dari suatu
sel.