Biochem E2: Todd Miller

Post-translational modifications

covalent modifications to the protein that takes place either during or after assembly on the ribosome, often alters the chemical nature of a sidechain

PTMs that occur frequently

removal of initiating Met residue removed, N-terminal acetylation

PTMs that do NOT occur frequently

hydroxylation of certain Pro and Lys residues is specific to procollagen, only about 0.1% of cellular phosphoproteins contain phosphotyrosine

Non-enzymatic PTMs

tend to occur to all susceptible residues, depends only on chemical environment, slow, examples are oxidation of Met or Cys or nitration of Tyr

Reversibility of PTMs

some are reversible, some are not

Irrreversible PTMs

acetylation of N-terminus or proteolysis

reversible PTMs

methylation

Specificity of PTMs

some are specific, others are not, consensus sequences

Consensus sequence

short regions of sequence that are recognized by a modifying enzyme, presence does not guarantee that the modification occurs

How would you demonstrate that a particular modification occurs in vitro?

in vitro reaction with pure proteins (kinase, protein, ATP and measure phosphorylation with SDS-PAGE), synthetic peptide experiments (enzyme, peptide gives modified peptide), reversal by chemical or enzymatic means

How would you demonstrate that a particular modification occurs in vivo?

incorporate radioactive precursor (treat cells with 32P and all phosphorylated proteins will be tagged with radioactive label, isolate protein, digest with a proteolytic enzyme, mass spec to map sites), inhibitor studies, mutational analysis

Most convincing methodology to prove PTM in vivo

combination: site of cellular modification mapped after incorporation of radioactive precursor, next mutant form of the protein lacking site of mod could be introduced to cells with radioactive precursor, SDS-PAGE analysis of cell extracts to show mutant cant be modified

Localization in PTMs

some modifications only occur in certain cellular compartments, example several modifications important for protein folding occur in the ER

Physiological importance of PTMs

some modifications are important while others do not appear to serve biological functions

Changes in activity of target protein due to PTMs

change in conformation (phosphorylase) , localization (farnesylation of Ras to bring it plasma membrane), chemical property of side chain (carboxylation of Glu)

Multisite modification

complex patterns of modification can act as a molecular barcode and can have additive or antagonistic effects

Additive Effects

when multiple modifications work in concert to amplify or stabilize a biological outcome, increases the binding affinity for an effector protein or progressively shifts the conformational state of a macromolecule

Antagonistic Effects

presence of one modification actively inhibits, blocks, or reverses the functional consequences of another through direct competition at a residue, allosteric or steric hinderance, functional opposition

Modifcation-dependent interactions with protein domains

can promote protein-protein interactions, some pro-pro interactions will only occur when a PTM is present, example is phosphorylation-dependent interactions in signal transduction

Proteolytic Processing

mainly proteins destined for cellular organelles or secretion, often a method to regulate biological activity of a protein (proprotein precursors), proproteins are often synthesized as preproproteins with an N-terminal signal peptide first that is removed in the ER during biosynthesis

Are proteases specific and why?

very specific, to avoid random proteolysis

Peptide hormone precursors

most peptide hormones are synthesized as larger precursors that are processed before secretion, example is insulin

Differential processing of POMC precursor

many hormone precursors contain several active peptide hormones, synthesis as a single precursor helps to coordinate action of hormones, different proteolytic clips can produce different combinations of hormones

Activation of proteolytic enzymes

zymogens to proteases, made in pancreas and processed to active proteases in the small intestine

Notch Receptor Protein

signaling dependent on regulated proteolysis, Notch is processed and activated by proteolytic cleavage

Signaling Pathways that use proteolysis

Notch, Wnt/B-catenin, NF-kB

Alterations of the N-terminus

deformylation of Met, N-terminal acetylation, removal of N-terminal Met, addition of terminal residues

Deformylation of Met

initiating Met residue on proteins synthesized in prokaryotesis formylated. this formyl group is removed by a deformylase.

N-terminal acetylation

catalyzed by ribosome-associated acetyltransferases, can occur without the Met, small residues preferred (Gly, Ala, Ser)

Biological role of N-terminal acetylation

regulation of protein stability, regulates some protein-protein interactions

Removal of N-terminal Met

removed by ribosome-associated Met-aminopeptidase, small/uncharged residues at the second residue

Addition of terminal residues

transfer of residues from charged tRNAs directly to N-termini, Leu/Phe put on proteins with an N-terminal Arg or Lys get targeted for degradation

Carboxylation of Glutamate

proteins involved in blood clotting and bone structure, enzyme responsible is an integral membrane protein in the ER, Gla residues bind Ca more tightly

Hydroxylation of Pro and Asn in HIF1a

Hypoxic conditions lead to constitutive expression of HIF1a which facilitates tumor spread. well oxygenated cells hydroxylate HIF1a to destroy it

Lysine Acetylation

reversible, histones, acetylation enhanced in the chromatin of active genes

Covalent modifications of histones

Lys acetylation, Lys methylation, Ser phosphorylation

Disulfide bond formation

occurs in lumen of ER, more oxidizing redox state than in cytosol, liked with 3D folding

Protein disulfide isomerase

assists in disulfide bond formation and isomerization

ADP-ribosylation

transfers an ADP-ribose group to target protein, cholera toxin and PARP1

Protein phosphorylation

activity of proteins are controlled after translation, regulation of metabolism, hormone action, cell growth, phosphate monoester possesses a -2 charge

Cholera Toxin

ADP-ribosylates the a-subunit of G-protein Gs, unable to hydrolyze GTP so stuck in active state

PARP1

involved in DNA repair and replication, gets ADP-ribosylated, PARP1 inhibitors for many cancers especially those linked to BRCA genes

Types of protein kinases

tyr kinases, ser/thr kinases, about 500 protein kinases in human genome, share conserved catalytic core of about 270 aa, high homology

CheA

histidine kinase, involved in bacterial chemotaxis, CheA phosphorylates itself on his, transfers its high energy phosphate to CheY, flagellar motion

Kinase consensus sequences

substrates share a set of common sequences that are necessary and sufficient for recognition, existence of apparent consensus sequence does not guarantee phosphorylation, for some kinases this sequence is very important while others rely on localization to play a key role

Phosphatase specificity

fairly nonspecific, regulated primarily by localization and substrate targeting

Phosphatase

Removes a phosphate group from a target substrate

How are consensus sequences found?

comparison of sequences around known phosphorylation sites to look for patterns, synthetic peptide studies where one aa is varied near the phosphoacceptor site and measue effect on Km and Vmax, use synthetic peptide libraries to screen kinase specificity

what are the two main mechanisms by which specificity of kinases and phosphatases are regulated?

protein domain on the enzyme or substrate, associated targeting subunit

Targeting via Domans: Receptor Kinases/Phosphatases

insulin receptor kinase, transmembrane region that anchors them in the membrane

Targeting via Domans: Tyrosine Phosphatase PTP1B

C-terminal 35 aa motif that directs it to the cytoplasmic face of the ER

Targeting via Domans: Tyrosine Phosphatase PTPMEG1

motif similar to cytoskeletal-associated proteins, plays a role in dephosphorylating cytoskeletal elements

Targeting via Domans: SH2, SH3 and modular domains

several kinases/phosphatases or substrates contain SH2, SH3 or other modular domains that mediate protein-protein interactions

Modular Signaling Domains

critical in regulating signaling pathways, organize protein-protein interactions

General features of signaling domains

fold into distinct structural units, compact, found in otherwise unrelated proteins, sequence conservation, fold and are functional when removed from parent sequence, 50-100 aa

General specificity of signaling domains

different domains bind to different ligands, specificity for particular target proteins arises from subtle variations within binding sites

Interaction of PLCy with the activated EGF receptor kinase

phosphorylation of PLCy by EGF receptor kinase is driven by SH2-dependent targeting

Targeting by associated subunits

a separate polypeptide can target a kinase/phosphatase to a specific organelle, membrane, or substrate, activity of the catalytic subunit may be allosterically regulated by the targeting subunit

A-kinase anchoring proteins (AKAPs)

functionally and structurally diverse family of scaffold proteins that bind to regulatory subunits of protein kinase A (PKA) and tether these enzymes to specific intracellular locations,

Activation of PKA

exists as a tetramer of 2 regulatory and 2 catalytic subunits when inactive, binding of cAMP to regulatory subunits dissociates them and activates the catalytic subunits

Cyclin-dependent protein kinases (Cdks)

regulate cell division cycle, heterodimers composed of catalytic subunit and a cyclin which acts as a regulatory subunit and is essential for activity, different cyclins enable catalytic subunits to phosphorylate different substrates at appropriate stages of the cell cycle

Structural features of protein kinase

well conserved, Lys residue is present in all protein kinases and is important in binding ATP

Protein Kinase autophosphorylation

ser/thr or tyr kinases, occurs when one kinase domain in a dimer phosphorylates the other, activates

Activation of insulin receptor kinase

autophosphorylation induces a conformational change leading to activation

Abl kinase

non-receptor kinase, encoded by philadelphia chromosome, a hyper-activated form of Abl leads to CML, gleevec is an inhibitor

Mechanism of Action: Gleevec

binds specifically to the unphosphorylated form of Abl and prevents the activation of it

Protein Tyrosine Phosphatases (PTPs)

most have associated domains that enhance specificity

Protein kinase autoinhibition

enzymes fold into their non-active form through interactions between the catalytic site and another site, allosteric activators cause conformational change that disrupts the association, sometimes autoinhibitory domain contain pseudosubstrate sequences

Experimental approaches to show kinase autoinhibition

autoinhibitory/regulatory domain