BIOL2200

• Proteins


  Cells' functional units
• Protein assembly


  1. Protein sequences encoded by genes (DNA)

  2. Translated from mRNA exported from nucleus

  3. Transcriptional and translational complexes are examples of cellular machines

  
• Protein structure


  • Determined by primary sequence
  • Folding aided by chaperones
  • Dimerization/oligomerization gives rise to quaternary structures
  • Structural motifs are combinations of secondary structures

  
• Structural motifs


  • Ring-finger motif
  • Zinc-finger motif
• Domains


  • Distinct regions of tertiary protein structure
  • Larger than structural motifs, in addition to structural properties they can also be functional (ie. enzymatic/catalytic activity)
  • Proteins are modular in this way
• Protein size/mass units


  • Dalton: standard unit used to measure mass at molecular or atomic scale
  • Protein generally measured in kilodaltons (kDa)
  • Protein complexes or machines in the megadaltons (MDa)
  • Svedberg unit (Sv or S): rate (velocity) of sedimentation on a centrifuge (sedimentation co-efficient)
• Quaternary vs Macromolecular Complex

  • Quaternary: comprising multiple different protein components (subunits)
  • Macromolecular Complex: typically very large, many examples >1 megadalton (MDa), 30-300 nm and visible under electron microscopy

  
• Assembly of macromolecular complexes

  1. Diverse modes of assembly

  2. Assembly around a core subunit/complex

  3. Formation of subcomplexes prior to assembly
• Functions of subunits in cellular machines

  • Target recognition
  • Target binding
  • Scaffolds
  • Linkers
  • Adaptors
  • Structural
  • Biomechanical
  • Catalytic/enzymatic
  • Mediator
  • Regulation (activation/inhibition)
• Advantages of multi-subunit complexes

  • Target recognition/binding
  • Scaffolding/linking
  • Structural
  • Catalytic/enzymatic
  • Mediator
  • Activity regulation (activation/inhibition)
  • Flexibility in modular subunits
  • Facilitates complexity and diversity in function and regulation
• ATP


  • Energy currency of the cell
  • Energy is released from ATP when the terminal phosphate bond is broken
• Post-translational Modifications (PTM)


  • Phosphorylation: addition of phosphate to tyrosine/serine/threonine/histidine residues
  • Ubiquitination: addition of one (monoubiquitination) or more (polyubiquitination) ubiquitin to lysine residues
  • PTMs change protein structure with varied consequences like changed activity, target for degradation, changed cellular location, changed structure or organization
• Allosteric Regulation


  • Change in protein structure/function due to non-covalent binding by a ligand (eg. calcium, nucleotides, another protein)
  • Ca2+/calmodulin switch: Ca2+ binding changes calmodulin structure to allow binding to target protein
  • GTPase switch: GTP binding changes protein structure to increase enzymatic activity

  
• Cellular membranes


  • Plasma membrane: two-layered shell of phospholipids
  • Internal membranes contain specialised protein machines and associated functions
• Transcription


  Transcription initiation complex: ~30 protein subunits complex with polymerases, linkers, adaptors, activators and DNA binding proteins (Transcription Factors)

  
• Translation


  1. Ribosome: machine to synthesize polypeptides from mRNA, made up of ribosomal RNA (rRNA) and proteins (ribonucleoprotein)

  2. Other components include messenger RNA (mRNA), transfer RNA (RNA) and nascent polypeptide

  3. Protein synthesis requires ATP
• Protein Folding


  1. Chaperonins: barrel-shaped folding machine (GroEL, 14 subunits total)

  2. Folding of polypeptides occur within the barrel (folding chamber)

  3. Process is driven by ATP hydrolysis
• Protein Transport


  Kinesin: microtubule motor required for cellular transport, 380 kDa heterotetramer, ATP binding and hydrolysis by the motor domains drives the "walking motion"
• Protein Degradation


  1. Proteasome: 2.5 MDa, 31 subunit molecular machine for controlled proteolysis, 20S catalytic barrel core, 2 x 19S regulatory subunits

  2. Ubiquitin (Ub) chains recognized by 19S regulatory subunit, polyubiquinitated proteins unfolded and degraded in 20S core in ATP-dependent manner

  
• Cellular machines are involved in the entire life cycle of a protein: synthesis, folding, transport, degradation
• Nucleus


  Large organelle (easily visible under light microscope), The "brain" or "hard drive" of the cell, DNA within encodes the majority (but not all!) proteins within a cell, Gene expression (transcription) determines the nature of the cell/organism, Complex organization: nuclear envelope, nucleolus, and nuclear pore, Highly dynamic (eg. dramatic restructure for mitosis)

  
• Nuclear Membrane


  • The nucleus is surrounded by two membranes + nuclear lamina = nuclear envelope, Inner nuclear membrane (INM) defines nucleus, Outer nuclear membrane (ONM) continuous with rough endoplasmic reticulum, INM and ONM are each phospholipid bilayers separated by perinuclear space

  
• Nuclear Lamina


  • Meshwork of filaments located adjacent to the inside face of the inner nuclear membrane, Comprised of a class of cytoskeletal proteins known as intermediate filaments (nuclear Lamins), Cage or lattice-like network that interconnects with nuclear pores, Provides nucleus with structure

  
• Nucleolus


  A sub-organelle and clearly defined structure within the nucleoplasm, Site of ribosome biogenesis, Formed around regions of DNA encoding ribosomal RNA (rRNA), Specifically tandem repeated clusters of rRNA genes – Nucleolar Organizer Regions (NOR), Hotspot of transcriptional activity (~80% of total RNA in cell is rRNA), Genetically defined structures formed as a result of making ribosomes
• Nuclear Bodies


  • Membraneless nuclear sub-compartments, Highly dynamic in nature and in constant flux, Concentrated regions of protein and RNA (ribonucleoprotein complexes), Formation associated with transcriptional and RNA processing activity, May enhance process efficiency and facilitate regulation
• Chromatin


  DNA+histone complex: Nucleosome, Packaging of over 2 m of DNA within nucleus, Chromatin structure is dynamic (extended/condensed), Chromatin structure determines gene expression

  
• Histone Post-Translational Modifications


  Unacetylated: chromatin is highly condensed (transcriptionally inactive) – HETEROCHROMATIN, Acetylation – chromatin is less condensed, (transcriptionally active) – EUCHROMATIN, Histone PTMs represents a "histone code" to determine gene expression, Proteins that modify histones control chromatin structure and access of DNA to replication, transcriptional and repair machinery

  
• Transcriptional Machinery


  Transcriptional activators bind to DNA to recruit chromatin remodeling complexes to "open up" chromatin structure, They also recruit a protein bridge (mediator) to help recruit transcription factors to a promoter sequence, Mediator complex facilitates assembly of the preinitiation complex that includes loading a RNA polymerase (RNA pol II) on DNA, Transcription is paused by an elongation factor complex (NELF/DSIF), Elongation pause is relieved by phosphorylation and remodeling of the elongation factors by a cdk/cyclin pair (P-TEFb)

  
• Ribosome biogenesis


  Ribosomal RNA is first transcribed by RNA Pol I as a large transcript (pre-rRNA) that is then processed to 28S, 18S and 5.8S mature rRNA found in ribosomes, 5S transcribed in nucleoplasm by RNA Pol III and diffuses into nucleolus, 60S and 40S ribosomal subunits undergo a quality control check prior to export into cytoplasm, Passage is through nuclear pores and mediated by nuclear export adaptors, Final assembly into functional translation machinery (80S) occurs in cytoplasm
• Nuclear Pore Complex (NPC)

  • Spans both nuclear membranes, Sole gateway in or out of nucleus, Allows passive diffusion by small molecules (<40 kDA), Anything bigger needs to be guided through, Pore is big enough to fit the larger of the ribosomal subunits (60S, ~25 nm), Human NPCs are truly gigantic ~125 MDa in size, Yet only made up of ~ 30 different proteins termed nucleoporins (Nups), Different Nups are repeated 8-, 16-, 32- or 64-times, 8-fold spoke symmetry to NPC structure, Cytoplasmic and nuclear asymmetry, Central channel is flexible, Scaffold, linker and transmembrane (anchoring) Nups, Barrier and transport Nups (FG-Nups) in cytoplasmic filaments, central channel and nuclear basket facilitate high-specificity binding of transporters and their cargos

  
• Nuclear Transport: Getting in and out of the nucleus

  Localization signal on the cargo (Nuclear Localization Sequence (NLS) for import, Nuclear Export Sequence (NES) for export), Delivery/Transporter System (Importins for nuclear import, Exportins for nuclear export), Control system for delivery utilizes a GTPase switch (Ran-GTP/GDP)
• Nuclear Transport: Ran-GTP gradient


  Drives unidirectional movement of cargo
• Laminopathies: Genetic mutations that impact lamins, nuclear membrane proteins connected to lamins or proteins involved in processing or maturation of lamins
• Laminopathies


  • Muscular Dystrophies and cardiomyopathies
  • Familial Partial Lipodystrophy
  • Peripheral and sensory neuropathies
  • Premature Aging (Progeria)
• Hutchison-Gilford Progeria Syndrome (HGPS): 1 in 10 million births, Condition of accelerated aging, Premature hair loss, muscle wasting, fat loss, reduced bone density, growth retardation, Death in mid-teens from heart failure or stroke, Mutations cause accumulation of unprocessed (premature) lamins (pre-lamins) or misprocessed lamins ("progerins")
• There's a little progerin in all of us, Accumulates with age, HGPS individuals have high levels of progerin at birth, Causes nuclear and genome instability (misshapen nuclei, chromatin disorganization, unrepaired DNA damage)
• Nuclear transport essential to link the process of transcription and translation which are separated by nuclear membrane
• Golgi Apparatus


  A basket or ribbon-like organelle located at the perinuclear region
• Golgi Apparatus


  • Organized around the centrosome/Microtubule Organizing Centre (MTOC)
  • Distinct from nuclear, ER and plasma membranes
• Golgi discovered in nerve cells by Camillo Golgi using a silver staining method


  1898