Cell and Molecular Biology

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Cell and Molecular Biology

• MSc Toxicology 2004
• Lecture 1
• 1) The dynamic cell
• 2) Proteins as a target for toxins

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The 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
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Structure 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

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All 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.

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Moving 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.

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Q 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.

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Why 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

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Proteins are made of amino acids
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Linkage 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

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The 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.

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Secondary 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.

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Domains

• 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
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Proteins 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
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Jobs 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

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Working 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.

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Binding 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.

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The 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
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Inhibitors

• 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.

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Natural 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

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A 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
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More 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.

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How 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

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Good 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