Lecture 2 Flashcards
Enzymes involved in synthesis or
degradation of second messengers
• These enzyme effectors can rapidly form second messengers from precursors or degrade second messengers by enzymatic reactions
• Second messengers are intracellular signaling molecules and can be:
– Diffusible small intracellular messengers like cAMP and IP3
– Membrane associated lipid messengers like DAG
• The second messengers activates and recruits cognate effector enzymes or transcription factors for further signal transduction
• Allow amplification of the signals as one effector molecule can syntesize many second messenger molecules
• We will look at two second messenger producing effector enzymes
– Phospholipase C (PLC)
– Adenylyl cyclase (AC)
PLC
• Different forms of PLC which can be activated by many different receptors including GPCRs and RTKs
• PLC is a membrane-associated enzyme – PH domain
• Lipase domain catalyzes the hydrolysis of the phospholipid phosphatidylinositol 4,5- bisphosphate (PIP2)
• Resulting in formation of two second messengers soluble inositol 1,4,5-
trisphosphate (IP3) and the membrane bound diacyl glycerol (DAG)
AC
• AC is a membrane-bound enzyme with two catalytic domains and two
integral membrane domains, each of which contains 6 transmembrane alpha-helices
• Adenylyl cyclase (AC) synthesize the second messenger cyclic adenosine monophosphate (cAMP)
• While phosphodiesterases (PDEs) degrade cAMP
Second messengers
• Intracellular signaling molecules
• Four different types of second messengers
– Lipids and lipid derivatives; like IP3 and DAG
– Cyclic nucleotides; cAMP and cGMP
– Ions; like Ca2+
– Gases; like Nitric oxide (NO)
• Most are soluble and diffusible, but some like DAG are membrane associated
• Some are like we have seen formed by enzymatic reactions performed by signal transduction effectors (PLC, AC)
• Others like Ca2+ is regulated by rapid release or uptake from/into
intracellular stores and/or efflux or uptake from the extracellular space
• Allow amplification of the signals
• Second messengers activates and recruits enzymes for further signal
transduction
Second messengers definition
Intracellular molecule mediates cell activity by relaying a signal initiated by an extracellular molecule (first messenger) activating a receptor.
+ amplfication
Kinase and phosphatase enzymes
• Another important group of signalling enzymes (effectors) are kinases
and phosphatases
• Protein Kinase: enzyme that catalyzes the transfer of a phosphate
group from one donor (usually ATP) to a protein
• Protein phosphatase: enzyme that catalyzes the hydrolysis (removal) of
a phosphate group from a protein
• Phophorylation of proteins (on serine, threonine or tyrosine
residues) is hence reversible and is used to regulate the activity of
proteins
• Phosphorylation results in the addition of a large negatively charged functional group and often will significantly affect the structure and function of the protein
• Phosphorylation can regulate;
– The activity
– Affinity (create docking sites)
– Localization of proteins
Transcription factors (TFs)
• The ultimate target of signaling pathways are often transcription
factors/transcription regulatory proteins that regulate gene expression and eventually allow transforming the recieved signal into a change of cellular activity.
• Transcription factors are proteins that bind to specific DNA
promoter sequences and activate or repress the transcription of
mRNA from that gene
• The DNA is packed into chromatin and the tightness of this packaging and thus how easily TFs can access the promoter can also be regulated by
various different complexes
• Signaling dependent TFs can be classified into;
– Nuclear receptor family
– TFs activated by internal signals (p53)
– Cell-surface receptor activated TFs
• CREB
• Notch intracellular domain (NICD)
Mouse
Pros
On average, the protein-coding regions of the mouse and human genomes are 85 percent identical
• Most signaling pathways are conserved
• Genetic tools been available a long time (transgene, knock-out or knock in)
• Well-established experimental model
• Lot of studies to compare with
• Inbred and disengaged from natural environment
• Used to study signaling in many processes
• Development of nervous system
• LTP, learning and memory
• Rearing of offspring
Cons
• We do not have complete map of neuronal connections in the mouse
nervous system
• Mouse life span ca 1-2 years
Species differences: Genetic and physiological differences may limit the translation of results to humans.
Limited complexity: Mice do not fully replicate human cognition, emotions, or complex behavior.
Inconsistent disease models: Some human diseases do not progress the same way in mice, and spontaneous diseases are often absent.
Ethical concerns: Issues with animal welfare and the ethics of genetic manipulation.
Cost and resources: Expensive housing, breeding, and long research timelines.
Immune system differences: Mice have a different immune system, which can affect disease models and therapy testing.
Limited translatability in drug development: Mice may metabolize drugs differently, leading to inaccurate predictions of drug safety and efficacy in humans.
Reproducibility issues: Variability in mouse models can complicate research findings.
Drosophila
Cons
* Simple nervous system and limited ability to model complex human brain functions and behaviors.
* Lack of key human organs like lungs, liver, and kidneys, limiting its use in modeling diseases involving these organs.
* No adaptive immune system, making it unsuitable for studying diseases that depend on adaptive immunity (e.g., autoimmune diseases, chronic infections).
* Inapplicability to some human diseases like advanced cancer, neurodegeneration, and certain complex genetic diseases.
* Limited social and cognitive complexity, making it less suitable for studying complex human psychological and psychiatric disorders.
* Short lifespan, which makes it difficult to study aging-related diseases or long-term health effects.
* Differences in metabolism, which hinder its use in human drug testing and pharmacokinetic studies.
Pros
• Life span ca 50 days
• Can be readily reared in the laboratory, breeds quickly, and lays many eggs
• Four pairs of chromosomes and good genetic tools available
• Many signaling pathways are relatively conserved
• Used to study signaling in many processes
• Development of nervous system
• Associate learning
• Movement, fighting
• Neurodegenerative diseases
• Well known anatomical situation
• Wiring diagram of 140,000 neurons and 54.5 million synapses in the drosophila brain published this year
Elegans
pros
Transparant
• Ca 20 day life span
• Around 60-80% of human genes have
c.elegans orthologs
• Unbiased genetic screens
• Used to study signaling in many processes
• Programmed cell death
• Development of nervous system
• Neurodegenerative diseases
• Role of neuropeptides in non-associative and associate learning
• Neuropeptides
• 302 neurons and all synapses mapped
• The major neurotransmitter systems are conserved
• The nerve ring ( purple) is the main central nervous system
– Receives sensory input from the amphids at the anterior tip of the animal.
– Many sensory neurons terminate their primary axons in the nerve ring, and sensory
integration can occur
• Ventral nerve cord with motor neuron cell bodies
Cons
1 Limited complexity of the nervous system and behavior, making it unsuitable for modeling complex cognitive functions or higher-level psychiatric disorders.
2 Absence of major human organs such as the liver, kidneys, and lungs, which limits its use for studying organ-specific diseases and systemic disorders.
3 Lack of adaptive immune system, which restricts its ability to model diseases involving human-specific immune responses.
4 Short lifespan and differences in aging processes, which limits long-term studies, especially of aging-related diseases.
5 Inapplicability to complex human diseases, such as neurodegenerative diseases, cancer, and metabolic disorders, which involve intricate human-specific mechanisms.
6 Experimental variability and challenges related to developmental differences, genetic backgrounds, and the environment in which the worms are grown.
7 Ethical concerns related to genetic modification and experimental conditions.
Cell lines
Pros
Pioneer” research systems in most fields of biomedicine
• Easy and cheap to culture/expand and to manipulate/treat
• Pure population of cells, which is valuable since it provides a consistent
sample and reproducible results
• Bypass ethical concerns associated with the use of animal and human
tissue
• Human cell lines – human genetic setup
• American Type Culture Collection (ATCC) Cell Biology Collection has a
number of neural cell lines
– SH-SY5Y
– LUHMES
Cons
• However, immortalized cell lines are genetically manipulated
• Serial passage of cell lines can further cause genotypic and
phenotypic changes
• Limited biological variation
Ipsc
• Somatic cells (fibroblast) from a human person is converted into an induced pluripotent
stem cell that can then be sequentially exposed to differentiation cues to form neurons
or other types of cells
• Possible to take somatic cells for various humans with different genotypes (ie have a
disease causing mutation or combination of risk genes) and compare
• However, if no appropriate control(s) are included data obtained can be vary confusing
Differentiation to cell type of interest
Induced pluripotent stem cell (iPSC) derived neurons
• More complex to culture
• Require knowledge of differentiation requirements
• Reduced suceptibility to tranfection and genome editing
Organoids
Cons
• Organoids should largely be considered as a model system under development
– Variability
– Lack of widely accepted standardized protocols
• Limitations
– Features similar to early brain development
– Lack vascular cells and immune cells, such as microglia
– Limited maturation and circuit formation
• Lack of high-fidelity cell types
• Multi-brain region assembloids allow for interaction between distinct brain regions
Pros
Human-specific models that provide more accurate representations of human organ structure and function than animal models.
Better modeling of human diseases, including genetic conditions, neurodevelopmental disorders, and cancer.
Ethical alternative to animal models in drug testing and disease research.
Personalized medicine applications by using patient-derived organoids for drug screening and therapy development.
Advancement in drug testing, including toxicity screening, to better predict human responses.
Studying human organ development and stem cell differentiation in a 3D, tissue-like environment.
Improved insights into complex diseases, such as cancer, infectious diseases, and organ-specific toxicities.
Studying organ microenvironments, including cell-cell interactions, which are crucial for understanding diseases like cancer and autoimmune conditions.
