Title: Cell and Molecular Biology
1Cell and Molecular Biology
- MSc Toxicology 2004
- Lecture 1
- 1) The dynamic cell
- 2) Proteins as a target for toxins
2The Structure of Cells
- The interior of eukaryotic cells consists of
organised structures (nucleus, mitochondria,
lysosomes peroxisomes and,in some cells secretion
granules) and of systems of membranes (the
endoplasmic reticulum, the Golgi apparatus, the
endosome system and a variety of transport
vesicles) suspended in
a fluid (the cytosol) and contained within the
plasma membrane
3Structure is governed by function
Secretion granules in the pancreas contain
digestive enzymes which will be discharged into
the intestine and insulin secreting cells with
much smaller granules
- Smooth endoplasmic reticulum in liver cells
contains the protective drug metabolising enzymes
4All is not as it seems
- Electron micrographs such as the ones on the last
slide give the impression of a very static cell
interior. This is not the case. The interior of
the cell is rapidly changing with organelles and
membrane systems breaking and rejoining.
Furthermore there is active transport of proteins
between organelles, for example proteins which
will be secreted from the cell pass from one
compartment to another.
5Moving and touching cells
- During embryonic life cells may migrate
considerable distances from the site of
differentiation to their final destination This
migration may be observed in wound healing or in
white blood cells hunting down bacteria. - The interior of a cell is thus a complex and
dynamic structure and the effects of xenobiotics
(chemicals not normally present in the body) will
be equally complex even before we consider
inter-actions between different cells and
tissues.
6Q How does biochemistry differ from chemistry
- A
- All fundamental laws such as conservation of
mass, the laws of thermodynamics etc. are still
obeyed but - Many reactions which are strongly exothermic do
not occur spontaneously because the activation
energy is too great.
7Why start here
- We will start with a discussion of proteins
because, in the end, toxicity is generally due to
a foreign compound (a xenobiotic) interfering
directly or indirectly with the function of a
protein. There are exceptions such as detergents
which interact with lipids and direct mutagens
which interact with DNA but, by in large it is
interactions with proteins which is the critical
event
8Proteins are made of amino acids
9Linkage of amino acids to other cell components
- The amide group in asparagine and the hydroxyl
groups of serine and threonine can bind to
carbohydrates - The hydroxyl groups of serine, threonine and
tyrosine can be phosphorylated. This plays a
major role in the control of metabolism in the
cells - In a reducing environment two cysteine molecules
may be oxidised to form an S-S bond which
stabilises folding
10The Structure of Proteins
- Proteins consist of one or more polypeptide
chains. A polypeptide consists of a series of
amino acids joined ny peptide bonds. These
chains are folded in a very precise fashion and
if this folding is abolished than the protein
generally becomes non-functional.
11Secondary and tertiary structure
- Typically the three dimensional structure of a
protein is composed of a series of fairly rigid
sections strongly stabilised by hydrogen bonds
(mainly alpha-helix and beta pleated sheet) and
flexible zones. - The flexibility means that a molecule binding at
one point on the surface of the protein will
produce sympathetic changes at other points, in
particular in the sites which bind the substrate
and catalyse the reaction.
12Domains
- The term domain is used to describe an area of a
protein which is functionally or physically
distinct. but this clearly consists of two or
more domains. For example many DNA binding
proteins contain a sequence known as a zinc
finger) be prepared for silly names. The
presence of complex proteins and of repeated
domains suggest how proteins have evolved and
become more specialised. Another example would
be proteins which pass through a membrane, eg
some plasma membrane proteins will have
cytosolic, transmembrane and extracellular
domains.
This is a protein called src which is involved in
the control of cell division and hence in cncer.
Catalysis of the reaction involves the yellow
and orange domains, regulation the green and
blue. Joining regions are dark green
13Proteins may consist of more than one polypeptide
chain.
- Whereas some proteins have a single polypeptide
chain and functional domains others are made up
from several polypeptide chains. These may be
very similar as in haemoglobin or quite distinct
in function. The arrangement of the different
peptides is sometimes call the quaternary
structure
When antigen binds to the T cell receptor then a
series of changes occur. The cell must recognise
both MHC and the antigen, cytokines must be
release and in a cytotoxic T cell the
cytoskeleton must be re-organised. Thats the
reason for the complexity
14Jobs for proteins
- There are too many to list but a first division
may be into - Structural proteins which, in general, bind to
other proteins, nucleic acids and phospholipids - Proteins forming the cytoskeleton
- Proteins which bind to small molecules and which
catalyse chemical reactions (enzymes). - Transport proteins which carry across small
molecules across membranes. - Regulatory proteins whose job is to control the
action of other proteins, especially of other
proteins
15Working together
- Some proteins have additional groups such as
biotin or FAD attached while others need the
assistance of small molecules such as NAD
These co-enzyme play in important role in the
action of the enzyme. They are often complex
structures and are not manufactured by human
beings (ie they are vitamins) - Other proteins contain metal ions especially
iron. A much smaller number need copper,
manganese, zinc, or selenium-containing amino
acids. - Interference in the supply of these co-factors
will result in malfunction of the enzyme system
concerned. If this cannot be compensated then
toxic changes will develop.
16Binding and catalytic sites on proteins
- By definition, all enzymes must have binding
sites for their substrates. These may be very
simple as with non-specific phosphatases or very
complex with recognitions sites being different
from the catalytic sites. In general these
recognition sites are pockets in the protein
substructure with several non-adjacent sections
of the chain contributing to the binding.
17The mode of action of lysozyme
As can be seen catalysis of the reaction depends
on Glu35 and Asp 52 being in exactly the right
place. Hence any change which alters the folding
near the active site will result in loss of
activity
18Inhibitors
- As seen in the last slide the efficiency of
catalysis depends on the correct binding of the
substrate to the enzyme This may be disrupted
when - A compound resembling the substrate binds to the
recognition site but cannot be transformed by the
catalytic site. The inhibitor here behaves like
the classical dog in a manger. This is termed
competitive inhibition as the effect can be
overwhelmed by large amounts of substrate - Refolding due to binding at other points on the
proteins surface will not be affected by binding
of substrate, this type of inhibition is
non-competitive.
19Natural Regulators
- The refolding that occurs when molecules bind to
the surface of a protein has been exploited in
evolution in several ways - Enzymes at critical points in metabolic pathways
(usually the first irreversible step) have
regulator sites in addition to their catalytic
sites. For example phosphofructokinase is
inhibited by citrate. Hence when the Krebs cycle
is clogged sugars are not sent down the
glycolysis pathway but are converted to fat or
glycogen
20A more complex example
Here four amino acids share a common precursor.
Note that feedback is from the product to the
first enzyme of branch leading to each amino acid
but that there is also feedback to the initial
formation of aspartyl phosphate
21More regulation
- 2) Feedback inhibition is very useful for short
term control. Longer term regulation may be
achieved by adding highly charged groups,
normally phosphates, to the protein. This
mechanism is heavily used in signalling pathways.
In such a chain protein kinases (which add on
phosphates) are teamed with protein phosphatases
to decide, for example, whether a message should
be passed further down the chain. The effects of
mistakes (cancer) are so great that complex
checking systems have evolved.
22How does this relate to toxins
- Binding of a xenobiotic or its metabolite to a
protein may result in - Loss of function of that protein
- Create a false signal in the cells regulatory
system. This is especially dangerous with
proteins involved in growth control. - Interfere in the organisation of the cell
- Interfere in the message passing between the
different cells and tissues of the body - And lots more
- BUT The body has evolved to be tolerant of change
so in most cases with low doses the bodys own
defense systems can restore equilibrium
23Good and bad news and a lesson
- The bad news. Almost any interaction of
xenobiotics with proteins may produce toxic
effects - The good news. The body has evolved to cope
- The lesson Toxicologists cant possible know all
they need to know they need to know the broad
picture and how to look things up